pine monoterpenes and pine bark beetles a marriage
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Abstract Pine-feeding bark beetles (Coleoptera:
Scolytidae) interact chemically with their host
pines (Coniferales: Pinaceae) via the behavioral,
physiological, and biochemical effects of one class
of isoprenoids, the monoterpenes and their
derivatives. Pine monoterpenes occur in the
oleoresin and function as behaviorally active
kairomones for pine bark beetles and their
predators, presenting a classic example of tri-
trophic chemical communication. The monoterp-
enes are also essential co-attractants for pine bark
beetle aggregation pheromones. Ironically, pine
monoterpenes are also toxic physiologically to
bark beetles at high vapor concentrations and are
considered an important component of the de-
fense of pines. Research over the last 30 years has
demonstrated that some bark beetle aggregation
pheromones arise through oxygenation of mon-
oterpenes, linking pheromone biosynthesis to the
host pines. Over the last 10 years, however, sev-
eral frequently occurring oxygenated monoter-
pene pheromone components (e.g., ipsenol,
ipsdienol and frontalin) have also been shown to
arise through highly regulated de novo pathways
in the beetles (reviewed in Seybold and Tittiger,
2003). The most interesting nexus between these
insects and their plant hosts involves the late-
stage reactions in the monoterpenoid biosynthetic
pathway, during which isomeric dimethylallyl
diphosphate and isopentenyl diphosphate are
ultimately elaborated to stereospecific monot-
erpenes in the trees and to hydroxylated monot-
erpenes or bicyclic acetals in the insects. There is
signal stereospecificity in both production of and
response to the monoterpenoid aggregation
pheromones of bark beetles and in response
to the monoterpenes of the pines. In the
California fivespined ips, Ips paraconfusus, we
Dedicated to Professor David L. Wood on the occasionof his 75th birthday, January 8, 2006
S. J. Seybold (&) Æ D. P. W. Huber Æ J. C. LeeChemical Ecology of Forest Insects, USDA ForestService, Pacific Southwest Research Station, 720Olive Drive, Suite D, Davis, CA 95616, USAe-mail: sseybold@fs.fed.us
D. P. W. Huber Æ J. C. LeeDepartment of Entomology, University of California,Davis, CA 95616, USA
D. P. W. HuberEcosystem Science and Management Program,University of Northern British Columbia, 3333University Way, Prince George, BC V2N 4Z9,Canada
A. D. GravesDepartment of Entomology, University of Minnesota,St. Paul, MN 55108-6125, USA
J. BohlmannMichael Smith Laboratories, University of BritishColumbia, 321-2185 East Mall, Vancouver,BC V6T 1Z4, Canada
Phytochem Rev (2006) 5:143–178
DOI 10.1007/s11101-006-9002-8
123
Pine monoterpenes and pine bark beetles: a marriageof convenience for defense and chemical communication
Steven J. Seybold Æ Dezene P. W. Huber ÆJana C. Lee Æ Andrew D. Graves ÆJorg Bohlmann
Received: 27 May 2005 / Accepted: 6 April 2006 / Published online: 5 July 2006� Springer Science+Business Media B.V. 2006
have discovered a number of cytochome P450
genes that have expression patterns indicating
that they may be involved in detoxifying mono-
terpene secondary metabolites and/or biosynthe-
sizing pheromone components. Both processes
result in the production of oxygenated monot-
erpenes, likely with varying degrees of stereo-
specificity. A behavioral analysis of the
stereospecific response of I. paraconfusus to its
pheromone is providing new insights into the
development of an efficacious bait for the detec-
tion of this polyphagous insect in areas outside
the western United States. In contrast, a Eurasian
species that has arrived in California, the Medi-
terranean pine engraver, Orthotomicus (Ips) ero-
sus, utilizes both a monoterpenoid (ipsdienol) and
a hemiterpenoid (2-methyl-3-buten-2-ol) in its
pheromone blend. The stereospecificity of the
response of O. erosus to the monoterpenoid
appears to be the key factor to the improved
potency of the attractant bait for this invasive
species.
Keywords Aggregation pheromone Æ Behavior ÆBiosynthesis Æ Coleoptera Æ Host colonization ÆIpsdienol Æ Ips paraconfusus Æ Kairomone Æ2-Methyl-3-buten-2-ol Æ Monoterpene Æ Myrcene ÆOrthotomicus erosus Æ Pinus Æ P450 Æ Scolytidae
Introduction
Bark beetles (Coleoptera: Scolytidae) are a group
of subcortical insects that feed as larvae and
adults in the phloem of trees and woody shrubs
(Wood and Bright 1992). They are closely allied
with another group of beetles, ambrosia beetles,
which tunnel into the xylem and derive nutrition
from associated fungi. Together there are nearly
6,000 species of Scolytidae worldwide, forming
one of the most formidable groups of endophytic
parasites known to mankind. Although no defin-
itive estimates are available, it is likely that over
500 species of scolytids feed on pine trees in the
genus Pinus, which is probably the most species-
rich group of conifers in the world (Critchfield
and Little 1966; Mirov 1967; Price et al. 1998).
Pine bark beetles display a variety of micro-
habitat associations with pines that include
colonization of cones (Conophthorus spp.), twigs
and small branches (Pityophthorus spp.), upper
stem and large branches (Ips spp., Orthotomicus
spp., Pityogenes spp., Pityokteines spp.), main
stem (Dendroctonus spp., Ips spp., Hylurgops
spp.), and lower stem, root collar, and roots
(Dendroctonus spp., Hylurgus spp., Hylastes spp.,
Tomicus spp.) (Fig. 1). Ambrosia beetles
(Gnathotrichus spp., Trypodendron spp., and
Xyleborus spp.) colonize the sapwood of the
lower stem. Many of these species also colonize
broken portions of trees that have fallen to the
ground or stumps that remain after a tree has
been broken or cut. In addition to these spatial
patterns related to gross host anatomy, these
beetles also partition themselves temporally, with
certain genera (e.g., Dendroctonus, Ips) prefering
to colonize recently declining or even healthy
trees, whereas other genera prefer to colonize
trees in a more advanced state of biodeterioration
(e.g., Hylurgops or Hylastes, the so-called sour
cambium beetles).
Host colonization in pine bark beetles involves
visual (Strom et al. 1999, 2001), olfactory (DL
Wood 1972, 1982), and gustatory signals (McNee
et al. 2000, 2003), which in most species culmi-
nates in the aggregation of many individuals in
the phloem in discrete family units defined spa-
tially by galleries. Aggregation pheromones are
used to signal the mass attack of the beetles on
pines, allowing the insects to coordinate feeding
and mating in time and space (DL Wood 1982;
Seybold et al. 2000). The mating systems are
varied (Kirkendall 1983; Kirkendall et al. 1997).
For example, in Dendroctonus spp. the female
tunnels through the bark and initiates the con-
struction of a somewhat longitudinally oriented
gallery, where she is later joined by a male in a
monogynous mating system (Hopkins 1909). The
galleries are packed with frass, which is the dust
that results from boring activity, and consists of
phloem and xylem fragments as well as the feces
(Wood et al. 1966). In contrast, in Ips spp. the
male initiates the construction of a longitudinally
oriented gallery, where he is later joined by many
females in a polygynous mating system (Struble
and Hall 1955). Ips spp. push the frass out of the
galleries onto the bark surface, resulting in an
open gallery system. These galleries assume a
144 Phytochem Rev (2006) 5:143–178
123
Y- or stellate shape, with a single female in each
arm. Hypothetically, the intent of these gallery
shapes is to avoid intraspecific competition
among the resulting larvae that feed in the
phloem away from the egg gallery walls (Poland
and Borden 1994; Robins and Reid 1997).
The influence of monoterpenes on pine bark
beetles
The behavior and physiology of pine bark beetles
during dispersal and at the time of host coloni-
zation are largely governed by the interactions of
the beetles with monoterpenes (Fig. 2). The
relationship between the beetles and these
isoprenoids is quixotic, and may have both posi-
tive and negative consequences for survival and
reproduction (Table 1). Volatile monoterpenes
pervade pine forest airspaces throughout the
Northern Hemisphere (Tingey and Burns 1980;
Guenther et al. 1994; Holzinger et al. 2005a).
Kesselmeier and Staudt (1999) estimate that the
global carbon input for monoterpenes ranges
between 127 and 480 Tg C year)1. Monoterpene
flux data for pines has been derived from (1)
emissions measured around foliage (Litvak and
Monson 1998; Litvak et al. 1999; Niinemets et al.
2002) or individual small trees (Tingley et al.
1980; Juuti et al. 1990; Shao et al. 2001) and (2)
measurements taken in or above the forest
canopy (Schade et al. 1999; Schade and Gold-
stein 2003; Holzinger et al. 2005a; A Lee et al.
2005). The fluxes are increased by disturbances
(Juuti et al. 1990; Stromvall and Petersson 1991;
Schade and Goldstein 2003); by temperature
(Tingey et al. 1980, 1991; Juuti et al. 1990;
Charron et al. 1995; Shao et al. 2001); and by
humidity (Schade et al. 1999), leading to dy-
namic diurnal emission patterns (Schade and
Goldstein 2003; Holzinger et al. 2005b). Mono-
terpene fluxes above a mixed conifer forest
containing primarily ponderosa pine, Pinus
ponderosa Dougl. ex Laws., in California’s cen-
tral Sierra Nevada mountains have ranged sea-
sonally from 0.10 to 0.83 lmol m)2 h)1
(Holzinger et al. 2005a) with basal emission rates
at 30�C in May ranging from 0.05 to 0.38 mg C
m)2 h)1, depending on the species of monoter-
pene evaluated (Schade and Goldstein 2003).
Pine bark beetles are thought to generally
constrain their dispersal flights within the height
Conophthorus spp.
Pityophthorus spp.
Hylastes macer
Hylastes macerHylurgops porosusDendroctonus valens
Dendroctonus brevicomisDendroctonus ponderosaeHylurgops porosus
Ips paraconfususIps piniPityogenes carinulatus
Ips latidensPityokteines ornatus
Gnathotrichus retususTrypodendron lineatumXyleborus intrusus
Fig. 1 Spatialcolonization patterns ofponderosa pine, Pinusponderosa Dougl. exLaws., by bark andambrosia beetles(Coleoptera: Scolytidae)in the central SierraNevada of California.Host associations of thespecies are based onBright and Stark (1973)and SL Wood (1982). Thisfigure is based on agraphic developed by DLWood (University ofCalifornia at Berkeley)
Phytochem Rev (2006) 5:143–178 145
123
of the stem of their host trees (Gara and Vite
1962; Schmitz 1980, 1984; Schmitz et al. 1980,
1989; Safranyik et al. 1989, 1992, 2000; Byers
2000; Safranyik and Carroll 2006). A small
percentage of the population may disperse
above the forest canopy (Furniss and Furniss
1972; Safranyik et al. 1992; Safranyik and Car-
roll 2006). Thus, monoterpene emissions from
the woody portions of stems and branches are
more likely to permit focused host-location
behavior by pine bark beetles and are likely to
be more relevant to their colonization behavior
than emissions from foliage. However, very little
information appears to be available on
these woody emissions or they are presumed to
be low under ambient conditions (Schade and
Goldstein 2003). When woody tissues are dam-
aged on standing trees or on portions of cut and
fallen trees during mechanical disturbances
such as forest harvest and thinning operations,
total emissions of monoterpenes increase sub-
stantially (Stromvall and Petersson 1991; Schade
and Goldstein 2003). The three-dimensional
aligment of the dispersal space of the beetles
with the emerging awareness of the dynamic
pool of background monoterpenes in forests
has only begun to be explored (Byers et al.
2000).
Fig. 2 Behaviorally active isoprenoids for pine bark beetlesincluding myrcene (7-methyl-3-methylene-1,6-octadiene),terpinolene [1-methyl-4-(1-methylethylidene)-cyclohexene],c-terpinene [1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene],b-phellandrene [methyl-6-(1-methylethyl)-cyclohexene], a-pinene (2,6,6-trimethylbicyclo[3.1.1] hept-2-ene), b-pinene(6,6-dimethyl-2-methylenebicyclo[3.1.1] heptane), 3-carene
(trimethylbicyclo[4.1.0]hept-3-ene), ipsenol (2-methyl-6-methylene-7-octen-4-ol), ipsdienol (2-methyl-6-methy-lene-2,7-octadien-4-ol), cis-verbenol (cis-2,6,6-trimethylbi-cyclo[3.1.1]hept-2-en-4-ol) [optical rotations of cis-verbenoldesignated as measured in chloroform, enantiomers alsoreferred to as (1S,4S,5S)-()) and (1R,4R,5R)-(+) by someauthors], and 2-methyl-3-buten-2-ol
146 Phytochem Rev (2006) 5:143–178
123
Monoterpenes as attractive kairomones
for pine bark beetles
Within the dynamic aerial sea of monoterpenes
and other volatile organic compounds that
characterize pine ecosystems, some species of
dispersing adult pine bark beetles manage to
focus their olfactory system on specific monot-
erpenes that emanate from specific pines. In
these cases, monoterpenes function as essential
host attractants (kairomones) that enhance the
reproduction and survival of the beetles (re-
viewed in Seybold et al. 2000). Researchers have
tested the behavioral impact of monoterpenes by
placing them in discrete release devices (i.e.,
near-point sources) from which the monoterp-
enes elute on the order of 10 to 1,000 mg/day.
For example, when tested individually, (S)-())-b-
pinene, (R)-(+)-a-pinene, and (S)-(+)-3-carene
(Fig. 2) all attracted the red turpentine beetle,
Dendroctonus valens LeConte, to multiple fun-
nel traps in the mixed conifer forest of Califor-
nia’s central Sierra Nevada mountains (Hobson
et al. 1993). These authors also demonstrated
that the three monoterpenes were present in the
oleoresin of two of the pines colonized in this
area by D. valens, P. ponderosa, and sugar pine,
Pinus lambertiana Dougl. Other pine-infesting
bark beetles that respond in flight significantly to
monoterpenes alone include the mountain pine
beetle, Dendroctonus ponderosae Hopkins (to
c-terpinene) (Miller and Borden 2003); the
western pine engraver, Ips latidens (LeConte),
and the pine engraver, Ips pini (Say) (both to
b-phellandrene) (Miller et al. 1986; Miller and
Borden 1990a, b, 2000); and the pine shoot
beetle, Tomicus piniperda L. [to (R)-(+)-a-
pinene, (S)-())-a-pinene, (S)-(+)-3-carene, and
terpinolene] (Byers et al. 1985; Schroeder and
Eidmann 1987; Schroeder 1988; Schroeder and
Lindelow 1989; Byers 1992; Czokajlo and Teale
1999; Poland et al. 2003, 2004). Both sexes of the
southern pine beetle, Dendroctonus frontalis
Zimm., responded to increasing doses of
a-pinene relative to a solvent control in a labo-
ratory walking bioassay (McCarty et al. 1980),
but the response to a-pinene alone was not
confirmed with flight behavior in a controlled
field experiment (Payne et al. 1978).
Monoterpenes as pine bark beetle pheromone
co-attractants
Monoterpenes may also work in concert with
beetle-produced compounds to enhance the
responses to aggregation pheromones (Table 1,
Vite 1970). A research team led by DL Wood and
RM Silverstein first discovered this phenomenon
with the western pine beetle, Dendroctonus
brevicomis LeConte (Silverstein et al. 1968,
Bedard et al. 1969, 1970, 1980; Wood et al. 1969;
Silverstein 1970a, b; Wood 1970, 1972). Using a
benzene extract of the frass from unmated
females feeding in P. ponderosa, laboratory as-
says of the walking behavior of both sexes of
D. brevicomis revealed that the response to
female-produced exo-brevicomin was synergized
by a hydrocarbon fraction that was inactive alone
(Silverstein et al. 1968; Silverstein 1970a, b); one
of the synergistic components of the hydrocarbon
fraction was isolated and identified as myrcene
(Fig. 2) (Silverstein 1970a, b). Myrcene, which is
present in the host volatiles from oleoresin of
P. ponderosa (Hobson et al. 1993) and Coulter
pine, P. coulteri D. Don (Smith 2000), also acted
synergistically with exo-brevicomin to attract
both sexes of the beetle in flight (Bedard et al.
1969, 1970). The synergistic effect of myrcene was
less evident when the monoterpene was tested
with the binary mixture of exo-brevicomin and
male-produced frontalin (Bedard et al. 1980).
Nonetheless, further field tests with exo-brevico-
min, frontalin, and six monoterpenes (each pre-
sented individually in the experiments) confirmed
that the combination with myrcene elicited the
highest trap catches (Wood 1972; Bedard et al.
1980). Distilled oleoresin (turpentine, whose
chemical composition was unreported) enhanced
the flight response to exo-brevicomin and front-
alin to a greater extent than myrcene (Wood
1972; Bedard et al. 1980). In these tests the
release rate of synthetic myrcene alone was
equivalent to its release rate from the turpentine
[24 mg/day, Bedard et al. (1980)] or likely
exceeded its release rate from the turpentine
[96 mg/day vs. 48 mg/day, assuming a 10%
myrcene content of the turpentine, Wood (1972)].
In another study, freshly tapped oleoresin
from P. ponderosa was unattractive alone to
Phytochem Rev (2006) 5:143–178 147
123
Ta
ble
1A
crit
ica
lsu
rve
yo
fre
sea
rch
on
pin
e-f
ee
din
gb
ark
be
etl
es
an
dth
eir
ph
ysi
olo
gic
al
an
db
eh
av
iora
lre
spo
nse
sto
mo
no
terp
en
es
Sp
eci
es
Re
spo
nse
sto
sele
cte
dm
on
ote
rpe
ne
s
Co
no
ph
tho
rus
con
iper
da
(±)-
a-Pineneincreasesmaleflightresponse
toracemic
tra
ns-pityolin
oneoffourexperim
ents;individualmonoterpenes
ormixturesare
notattractiveto
malesorfemalesin
theabsence
ofpityol(D
eGrootet
al.1998);flightresponse
toa-pineneisdose-andenantiospecificwhen
combined
withracemic
tra
ns-pityol(M
illeret
al.2003).
Co
no
ph
tho
rus
po
nd
ero
sae
())-
a-Pineneincreasesmale
flightresponse
toracemic
tra
ns-pityol-baited
traps(M
illeret
al.2000).
Co
no
ph
tho
rus
resi
no
sae
(±)-
a-Pinene,())-
b-pinene,(+
)-a-limonene,andmyrcenein
variouscombinationsdid
notenhance
theflightresponse
ofmalesto
racemic
tra
ns-pityolin
aseries
ofthreeexperim
ents;thepinenes
werenotattractivealone(D
eGrootandZylstra1995)
Den
dro
cto
nu
sb
rev
ico
mis
Lim
on
en
eis
tox
ic(S
mit
h1
96
5a
,b
)a
nd
Pin
us
po
nd
ero
satr
ee
sfr
om
are
as
wit
hh
isto
rica
lly
hig
hp
op
ula
tio
ns
of
D.
bre
vic
om
ish
av
ere
lati
ve
lyh
igh
con
cen
tra
tio
ns
of
lim
on
en
e,
my
rce
ne
,a
nd
b-pinene,butlow
concentrationsofa-pinene(Sturgeon1979);myrcenemetabolizedbyadultsofboth
sexes
tomyrcenol(R
enwicket
al.1976b)andbymalesto
ipsdienol(R
enwicket
al.1976b;Byers1982;Seybold
etal.1992);camphenemetabolizedbyadults
ofboth
sexes
to6-hydroxy-camphen
e(R
enwicket
al.1976b);
a-pinenemetabolizedbyim
mature
(teneral)adultsto
tra
ns-verbenolatlower
ratesthanby
mature
adults;
a-pinenealsometabolizedbymature,butnotteneral,adultmalesto
verbenone(Byers1983b);myrceneenhancesthewalkingandflight
responsesto
thepheromonecomponent
exo-brevicomin
(Bedard
etal.1969,1970;Silverstein1970a,b)andtheflightresponse
toex
o-brevicomin
and
frontalin
(Wood
1972;Wood
etal.
1976;Bedard
etal.
1980).
Other
monoterpenes
(camphene,
3-carene,
limonene,
a-pinene,
and
b-pinene),
P.
po
nd
ero
saoleoresin,ordistilled
P.
po
nd
ero
saoleoresin(turpentine)
either
donotincrease
orweakly
increase
theflightresponse
toex
o-brevicomin
andfrontalin(V
iteandPitman1969;Wood1972;Bedard
etal.1980).
Den
dro
cto
nu
sfr
on
tali
sL
imo
ne
ne
,b-pinene,
a-pinene,andcampheneare
toxic,in
descendingorder
oftoxicity(C
ookandHain
1988);camphenemetabolizedbyadultsofboth
sexes
to6-hydroxy-camphen
e(R
enwicket
al.1976b);
a-pinenemetabolizedbylarvaeandadults,butnotbypupae,to
tra
ns-verbenolandbyadultmales
toverbenone(H
ughes
1975);
a-pinenemetabolizedto
tra
ns-verbenol,verbenone,
andmyrtenolbyteneraladult
females(t
ran
s-verbenolconversion
enhancedbymethoprenetreatm
ent)(Bridges
1982);antennalresponsesofboth
sexes
toa-pineneand3-carene(D
ickensandPayne1977);
Pin
us
taed
a
turpentineenhancesresponse
toaggregationpheromone(Billings1985);
a-pineneelicited
asignificantanddose-dependentresponse
from
both
sexes
ina
laboratory
walkingbioassay(M
cCartyet
al.1980),buttheadditiveeffect
ofa-pineneto
thepheromonecomponentfrontalinwasnottested
directlyin
theexperim
ent;
a-pinenemayormaynotenhance
theflightresponse
tofrontalinastheexperim
entalevidence
iseither
notanalyzedstatistically
(RenwickandVite1969),notpresent(Payneet
al.1978),orconfounded
bythepresence
ofturpentinein
theexperim
ent(Billings1985).
Den
dro
cto
nu
sp
on
der
osa
ea-Pinenemetabolized
bymature
femalesandmalesto
tra
ns-verbenol(H
ughes
1973b);
())-a-Pinenemetabolized
bymature
femalesto
verbenene,
p-m
entha-1,5,8-triene,and
o-and
p-cymene(deuteratedsubstrate
andproducts),())-,(+
)-,and(±
)-a-pinenemetabolizedbyfemalesto
tra
ns-verbenol
inadose-dependentmanner
(Gries
etal.1990);antennalresponsesto
a-pinene,
b-pinene,
myrcene,
(E)-ocimene,
b-phellandrene,
limonene,
camphene,
sabinene,3-carene,
a-terpinene,
p-cymene,
c-terpinene,andterpinolene(H
uber
etal.2000;Puresw
aranet
al.2004a);
c-terpineneattractiverelativeto
an
unbaited
trapin
fieldflightassay(M
illerandBorden
2003)andattractiveathighrelease
rates(~52and1,110mg/day)when
combined
with
exo-
brevicomin
and
tra
ns-and
cis-verbenol(=
aggregationpheromone)
(MillerandBorden
2000);
a-pinenecombined
with
tra
ns-verbenolfoundto
bemore
attractivethancampheneormyrcene,whichweremore
attractivethaneither
limonene,3-carene,orb-pinenein
anuncontrolled
flightassayin
whichthe
resultswerenotanalyzedstatistically(Pitman1971);myrceneandterpinolenefoundto
bemore
attractivewhen
combined
with
tra
ns-verbenolthanwere
3-carene,limonene,
a-pinene,or
b-pinene(Billingset
al.1976);myrcenefoundto
bemore
attractivethan
a-pineneandother
monoterpenes
inpresence
ofpheromonecomponents
(Borden
etal.1983;Connet
al.1983;MillerandLindgren2000);highrelease
ratesof3-carene(~600mg/day)(M
illerand
Borden
2000)and
myrcene(900–6,500mg/day)(Borden
etal.
1987;Millerand
Borden
2000)each
increased
flightresponse
totheaggregation
pheromone;
b-phellandreneelicited
adose-dependentandincreasingflightresponse
totheaggregationpheromone,
butnorelease
ratessignificantly
increasedtrapcatchrelativeto
thepheromonealone(M
illerandBorden
2000),however,itwasattractiveover
arangeofdosesin
combinationwith
ipsdienol(M
illerandBorden
1990a);myrcenesynergized
attractionto
aggregationpheromone,buttherewasnoprimary
attractiondetectedto
avariety
ofmixturesofmonoterpenoids(Puresw
aranandBorden
2005);myrcenemaybesuperfluousin
incitingattack
onstanding
Pin
us
con
tort
ala
tifo
liawith
tra
ns-verbenoland
exo-brevicomin
(Borden
etal.1990).
148 Phytochem Rev (2006) 5:143–178
123
Ta
ble
1co
nti
nu
ed
Sp
eci
es
Re
spo
nse
sto
sele
cte
dm
on
ote
rpe
ne
s
Den
dro
cto
nu
ste
reb
ran
sa-Pinenemetabolizedbylarvaeandadults,butnotbypupae,
totr
an
s-verbenol(H
ughes
1975);
a-pinenemetabolizedbymicrosomalfractionoflarvae
andadultsto
a-pineneoxide(W
hiteet
al.1979);dose-dependentantennalresponsesto
a-and
b-pineneand
P.
taed
aturpentine(D
elorm
eandPayne
1990);turpentineisattractivein
thefield(reviewed
inNationet
al.1996);ethanolsynergizes
theflightresponse
toturpentinefrom
Pin
us
elli
ott
iiel
lio
ttii
Engelm.and
P.p
alu
stri
sMill.(contained
a-and
b-pinene,camphene,limonene,
b-phellandrene,andmyrcene)
when
thetw
omaterialswerecombined
in
onesolution,butnotwhen
they
werereleasedfrom
individualdevices
(Phillipset
al.1988).
Den
dro
cto
nu
sv
ale
ns
a-Pinenemetabolized
bymale
adultsto
cis-
and
tra
ns-verbenoland
cis-3-pinen-2-ol,
b-pinenemetabolized
bymalesto
pinocarvoneand
tra
ns-
pinocarveol(H
ughes
1973a);antennalresponsesbyboth
sexes
to(R
)-(+
)-and(S)-())-a-pinene,
(S)-())-
b-pinene,
(R)-(+
)-and(S)-())-limonene,
(S)-
(+)-3-carene,
myrcene,
b-phellandrene,
andterpinolene(W
hiteandHobson1993).Responsesbyantennaeto
(R)-(+
)-and(S)-())-a-pinenesuggest
differentreceptors
foreach
enantiomer
(WhiteandHobson1993).(+
)-a-Pinene,
())-
b-pinene,
and3-careneare
attractivein
flightassaysofNorth
Americanpopulations(H
obsonet
al.1993;Fettiget
al.2004),but3-careneisthebestattractantin
introducedpopulationsin
China(Sunet
al.2004).
Highrelease
rate
of(+
)-a-pinene(estim
atedat12,375–16,500mg/day)isattractive,butflightresponse
totheattractant(12,375mg/day)isinterrupted
by())-a-pinene(4,125mg/day)(H
obsonet
al.1993);())-b-Pinenewithethanolwasmore
attractivethan())-
a-pinenealoneorwithethanol(Petrice
etal.2004);highrelease
rate
ofethanolincreasedtrapcatchto
(±)-
a-pineneand())-
b-pinene(Josephet
al.2001).
Gn
ath
otr
ich
us
retu
sus
a-Pineneisneither
ahostattractantalonenorasynergistoftheaggregationpheromonecomponentsulcatol(Borden
etal.1980,1981;Liu
andMcL
ean
1989);highrelease
rate
ofethanolincreasedtrapcatchto
(±)-
a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
last
esa
ng
ust
atu
sa-Pineneandmyrceneweretested
incombinationwithethanolin
choiceassayswith
Pin
us
pa
tula
bark,buttheexperim
entswerenotdesigned
totestfor
theeffect
ofthemonoterpenes
relativeto
either
ethanolorbark
alone(Erasm
usandChown1994).
Hy
last
esa
ter
b-Pinenewithethanol,andrawturpentinewithethanolincreasedtrapcapturesrelativeto
anunbaited
trap;a-pinene(w
ithorwithoutethanol)did
not
increase
trapcapturesrelativeto
anunbaited
trap(R
eayandWalsh2002).
Hy
last
esb
run
neu
s()
)-a-Pineneincreasesattractionto
ethanol(Schroeder
andLindelow
1989).
Hy
last
escu
nic
ula
ris
())-
a-Pineneincreasesattractionto
ethanol(Schroeder
andLindelow
1989).
Hy
last
eslo
ng
ico
llis
Att
ract
ed
tom
yrc
en
e,b-pinene,
terpinolene,
b-phellandrene,
and3-carene,
each
incombinationwithipsenol(M
illerandBorden
1990b);highrelease
rate
ofethanolincreasedtrapcatchto
(±)-
a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
last
esm
ace
rH
igh
rele
ase
rate
of
eth
an
ol
incr
ea
sed
tra
pca
tch
to(±
)-a-pineneand())-b-pinene(Josephet
al.2001).
Hy
last
esn
igri
nu
sA
ttra
cte
dto
a-pinene(W
itcoskyet
al.1987);highrelease
rate
ofethanolincreasedtrapcatchto
(±)-
a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
last
eso
pa
cus
Att
ract
ed
to()
)-a-pineneand())-a-pinenewithethanol(Schroeder
andLindelow
1989)orto
a-pinenealoneorto
b-pinenewithethanolrelativeto
a
trapbaited
with
Ips
typ
og
rap
hu
spheromone(Petrice
etal.2004).Attracted
more
tothecombinationofnonanaland())-a-pinenethanto
())-a-pinene
alone(D
eGrootandPoland2003).
Hy
last
essa
leb
rosu
sE
tha
no
lin
cre
ase
sfl
igh
tre
spo
nse
toP
.el
lio
ttii
/P.
pa
lust
ris
turp
en
tin
e(c
on
tain
ed
a-and
b-pinene,camphene,limonene,
b-phellandrene,andmyrcene).
Experim
entlacked
anunbaited
controlto
proveresponse
toturpentinealone(Phillips1990).
Hy
lurg
op
sp
all
iatu
sA
ttra
cte
dto
am
ixtu
reo
f3
-ca
ren
e,
(+)-
an
d()
)-a-pineneandterpinolenewithethanol,butnotto
themonoterpenemixture
aloneandonly
weakly
to
ethanolalone(Byers1992);notattracted
to())-a-pinenealone(Schroeder
1988)orattracted
toonerelease
rate
of())-
a-pinene(Schroeder
andLindelow
1989),
butattracted
to())-
a-pineneand
ethanol(Schroeder
1988,2003;Schroeder
and
Lindelow
1989);
attracted
tob-pineneor
b-pineneand
terpinolene,
butnotterpinolenealonewhen
thesecomponents
werecombined
withethanol(V
olz
1988);trapped
inresponse
toterpinoleneand(±
)-a-
pinene(individuallyandcombined)when
thesecomponents
werecombined
withethanol(nonegativecontrol)(V
iteet
al.1986).
Phytochem Rev (2006) 5:143–178 149
123
Ta
ble
1co
nti
nu
ed
Sp
eci
es
Re
spo
nse
sto
sele
cte
dm
on
ote
rpe
ne
s
Hy
lurg
op
sp
oro
sus
Att
ract
ed
tote
rpin
ole
ne
,b-phellandrene,
and3-carene,
each
incombinationwithipsenol(M
illerandBorden
1990b);
highrelease
rate
ofethanol
increasedtrapcatchto
(±)-
a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
lurg
op
sre
ticu
latu
sH
igh
rele
ase
rate
of
eth
an
ol
incr
ea
sed
tra
pca
tch
to(±
)-a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
lurg
op
ssu
bco
stu
latu
sH
igh
rele
ase
rate
of
eth
an
ol
incr
ea
sed
tra
pca
tch
to(±
)-a-pineneand())-
b-pinene(Josephet
al.2001).
Hy
lurg
us
lig
nip
erd
aa-Pinene,
b-pinene,
andraw
turpentinewhen
combined
withethanolincrease
trapcapturesrelativeto
anunbaited
trap;a-pinenealoneand
b-pinene
alonealsoincrease
trapcaptures(R
eayandWalsh2002);
a-orb-pinenewhen
combined
withethanolora-pinenealoneincrease
trapcapturesrelativeto
atrapbaited
with
Ips
typ
og
rap
hu
spheromone(Petrice
etal.2004).
Ips
av
uls
us
My
rce
ne
me
tab
oli
zed
by
ma
les
toip
sdie
no
l(H
ug
he
s1
97
4);
do
se-d
ep
en
de
nt
an
ten
na
lre
spo
nse
so
fb
oth
sex
es
toa-pinene(Smith
etal.
1988);
turpentinepresentedatahighrelease
rate
reducedtrapcatchto
ageneric
pheromonebaitfor
Ipsspp.(Billings1985).
Ips
call
igra
ph
us
Ca
mp
he
ne
,li
mo
ne
ne
,an
db-pineneare
toxicat40and100ppm;a-pineneistoxicat100ppm;limoneneistheleasttoxicandcampheneisthemosttoxic
ofthemonoterpenes
at100ppm
(CookandHain
1988);dose-dependentantennalresponsesofboth
sexes
toa-pinene(Smithet
al.1988).
Ips
gra
nd
ico
llis
Do
se-d
ep
en
de
nt
an
dsi
mil
ar
an
ten
na
lre
spo
nse
sb
yb
oth
sex
es
toa-pinene(Smithet
al.1988;Ascoli-C
hristensenet
al.1993);
a-pinene,
b-pinene,
myrcene,
limonene,
camphene,
andcareneare
attractants
(Werner
1972;ChenierandPhilogene1989;Erbilgin
andRaffa2000);camphene,
limonene,
andmyrceneappearedto
enhance
flightresponsesto
anextract
of
I.g
ran
dic
oll
isfrass,buttheresultswerenotanalyzedstatistically(W
erner
1972);
turpentinepresentedatahighrelease
rate
enhancedtrapcatchto
ageneric
pheromonebaitfor
Ipsspp.(Billings1985).
a-Pinenewhen
combined
with
ethanolor
a-pinenealoneincreasedtrapcapturesrelativeto
atrapbaited
with
Ips
typ
og
rap
hu
spheromone(Petrice
etal.2004).
Ips
lati
den
sA
tso
me
rele
ase
rate
sb-phellandrene(~200mg/day)and
b-pinene(~240–1,200mg/day)increase
attractionto
ipsenol,buthighrelease
rates(100–
2,000mg/day)ofa-pinene,
3-carene,
terpinolene,
andmyrcene,
andonerelease
rate
ofb-phellandrene(~2,100mg/day)interrupttheflightresponse
to
ipsenol(M
illerandBorden
1990b,2000).
Ips
mex
ica
nu
sA
ttra
cte
dto
b-phellandreneand3-carene,
each
incombinationwithipsenol(M
illerandBorden
1990b).
Ips
pa
raco
nfu
sus
3-C
are
ne
me
tab
oli
zed
by
ma
les
to1
-me
thy
l-5
-(a-hydroxy-isopropyl)-cyclohexa-1,3-diene(R
enwicket
al.1976b);myrcenemetabolizedbymature
adult
males,butnotfemalesorim
mature
(teneral)males,to
ipsdienolandipsenol(H
ughes
1974;Byerset
al.1979;Hendry
etal.1980;Byers1983b);
a-pinene
metabolized
toci
s-and
tra
ns-verbenolbymature
and
teneral,
adult
malesand
females(R
enwick
etal.
1976a;Byers1981,1983b);
())-
a-pinene
metabolizeddose-dependentlyto
cis-
and
tra
ns-verbenolandmyrtenolbymalesandfemales,
malesproduce
more
ofthesecompounds(Byers1981);
myrceneinducesa‘‘coma’’in
beetles
athighheadspace
concentrations(Byerset
al.1979);
a-pinenecausesmortality
athighheadspace
concentrations
(Byers1981);dose-dependentantennalresponsesto
myrceneand(+
)-a-pinene(Light1983).
Ips
pin
i3
-Ca
ren
em
eta
bo
lize
db
ym
ale
sto
1-m
eth
yl-
5-(
a-hydroxy-isopropyl)-cyclohexa-1,3-diene(R
enwicket
al.1976b);
myrcenemetabolizedto
ipsdienol
(Vanderwel
1991);
antennalresponsesto
b-phellandreneandlimonene(H
uber
etal.2000);
())-,(+
)-,and(±
)-a-pinene,
(±)-
b-pinene,
and(±
)-
limoneneatincreasingconcentrationsgenerallyinhibitpostlandingbehaviors
(initialgalleryentry,within
tissueorientation,andgalleryextension)by
males(W
allin
andRaffa2000);
b-phellandreneisweakly
attractivealonein
flightbehavioralassays(M
illerandBorden
1990a);varioushighrelease
rates
of3-carene(~200–1,200mg/day),
b-phellandrene(~5,40–1,000mg/day),and
b-pinene(~240–1,200mg/day)increase
flightresponse
toipsdienol(M
iller
andBorden
1990a,2000,2003);highrelease
rates(~200–340mg/day)ofmyrcene,
())-
and(+
)-a-pinene,
and())-b-pinenereduce
flightresponse
to
ipsdienolandlanierone(Erbilgin
andRaffa2000);(±
)-a-pineneelicitsadose-dependentflightresponse
from
both
sexes
when
combined
withipsdienol
and
lanierone,
attractiveat
~60mg/day,interruptiveat
~350mg/day(Erbilgin
etal.
2003);
high
release
ratesofmyrcene(~50–650mg/day)and
terpinolene(~340–2,100mg/day)interrupttheflightresponse
toipsdienol(M
illerandBorden
2000,2003).
150 Phytochem Rev (2006) 5:143–178
123
Ta
ble
1co
nti
nu
ed
Sp
eci
es
Re
spo
nse
sto
sele
cte
dm
on
ote
rpe
ne
s
Ips
steb
bin
gi
(=sc
hm
utz
enh
ofe
ri)
())-
a-Pinene[>
68.5%-(
))]wastested
incombinationwithdifferentconcentrationsof(±
)-ipsenolin
aflightbioassay.Because
oftheabsence
ofa
negativecontrol,therewasnodefinitiveproofofaresponse
toa-pinenealone.
Inoneexperim
ent,theresponse
toa-pineneandipsenoldid
notdiffer
from
theresponse
toa-pinenealone(K
ohnle
etal.1988).
Pit
yo
gen
esb
iden
tatu
s()
)-a-Pinene,(+
)-a-pinene,andterpinoleneathighreleaserates(144mg/day)interruptattractionto
cis-verbenolandgrandisol(El-Sayed
andByers2000).
())-a-Pinene(33.6
mg/day),(+
)-a-pinene(33.6
mg/day),())-b-Pinene(23.5
mg/day),(+
)-3-carene(51.1
mg/day)andterpinolene(18.7
mg/day)orthe
combinationof(±
)-a-pinene,(+
)-3-carene,andterpinolene(60mg/daytotalrelease)interruptattractionto
cis-verbenolandgrandisol(Byerset
al.2000).
Pit
yo
gen
esk
nec
hte
li3
-Ca
ren
ea
nd
())-
a-pineneinterruptattractionto
ipsdienol(M
illerandBorden
2003).
To
mic
us
min
or
An
ten
na
lre
spo
nse
sto
(+)-
a-pinene,
())-
a-pinene,
(+)-3-carene,
myrcene,
andterpinolene(Lanneet
al.1987).
To
mic
us
pin
iper
da
An
ten
na
lre
spo
nse
sto
(+)-
a-pinene,
())-
a-pinene,
(+)-3-carene,
myrcene,
andterpinolene(Lanneet
al.1987).Attracted
tovariouscombinationsof
3-carene,(±
)-a-pinene,(+
)-a-pinene,())-a-pinene,andterpinolenewithandwithoutethanol(Byerset
al.1985;Klimetzeket
al.1986;Viteet
al.1986;
Schroeder
andEidmann1987;Byers1992);attracted
to())-
a-pinene(Schroeder
1988;Schroeder
andLindelow1989;CzokajloandTeale1999;Poland
andHaack
2000;Polandet
al.2003);attracted
tob-pineneandterpinolenewhen
combined
withethanol(V
olz1988);attracted
toa1:7
mixture
of(+
)-a-
pineneandethanol(Zumr1989);to
1:0.1,1:0.9,and1:9
mixturesofracemic
a-pineneandethanol(C
zokajloandTeale1999);to
a1:10mixture
of())-a-
pineneandethanol(Schroeder
2003);andto
variouscombinationsofa-pinene,
b-pinene,andethanol(Petrice
etal.2004).Additionof
tra
ns-verbenolto
())-a-pinenealoneorto
theblendof())-
a-pineneandother
semiochem
icalswasattractivein
Michigan,USandOntario,Canada(Polandet
al.2003,
2004),butnotsignificantlymore
attractivethanahost
compoundblend(ethanol,
b-pinene,
terpinolene)
inGermany(V
olz
1988).
Try
po
den
dro
nli
nea
tum
())-
a-Pineneisnotattractivealone,butitincreasedattractionto
ethanol(Schroeder
1988;Schroeder
andLindelow1989),whereasin
another
studythe
combinationof(±
)-a-pinene(30mg/day)andethanol(120mg/day)wasnotattractive(Borden
etal.1981);
a-pineneandethanolcombined
enhance
the
response
tothepheromonecomponentlineatin(V
iteandBakke1979;Shore
andMcL
ean1983;PaivaandKiesel1985),atlow
release
rates(28.8–
33.6
mg/day)a-pinene(unreported
enantiomericcomposition)increasedtheresponse
tolineatinandethanol(12.0–14.4
mg/day),butathigher
a-pinene
release
rates(86.4–100.8
mg/day)theincrease
wasless
pronounced(Bakke1983).
())-
a-Pinenealone(413mg/day)elicited
significantlyhigher
trap
catches
thanmyrcenealone(281mg/day)(M
illerandLindgren2000).
Ass
oci
ati
on
so
fth
ese
sco
lyti
dsp
eci
es
wit
hP
inu
sh
av
eb
ee
nd
ocu
me
nte
din
Bri
gh
ta
nd
Sta
rk(1
97
3);
SL
Wo
od
(19
82
);W
oo
da
nd
Bri
gh
t(1
99
2);
an
dB
rig
ht
an
dS
kid
mo
re(2
00
2)
Phytochem Rev (2006) 5:143–178 151
123
D. brevicomis, but enhanced the flight response to
exo-brevicomin and frontalin three-fold (Vite and
Pitman 1969).
Other examples of the positive influence of
monoterpenes as co-attractants on the response to
aggregation pheromone include (1) the eastern
fivespined ips, Ips grandicollis (Eichhoff), and
camphene, limonene, or myrcene (Werner 1972),
a-pinene (Erbilgin and Raffa 2000), or turpentine
from loblolly pine, Pinus taeda L. (Billings 1985);
(2) I. pini and 3-carene, b-phellandrene, or
b-pinene (Miller and Borden 2000, 2003) or certain
release rates of a-pinene (Erbilgin et al. 2003); and
(3) D. ponderosae and a-pinene (Pitman 1971, but
see Table 1 about the quality of this experiment),
myrcene (Borden et al. 1983, 1987; Conn et al.
1983; Miller and Lindgren 2000; Pureswaran and
Borden 2005), myrcene or terpinolene (Billings
et al. 1976), or 3-carene, myrcene, or b-phelland-
rene (Miller and Borden 2000). The role of
a-pinene (Renwick and Vite 1969) as a co-attrac-
tant in the pheromone of D. frontalis is confounded
by laboratory experiments that have not tested
directly the comparative responses to frontalin
with and without the monoterpene (McCarty et al.
1980); by field experiments with a minor treatment
effect but no statistical analysis (Renwick and Vite
1969); by field experiments with no treatment ef-
fect related to a-pinene (Payne et al. 1978); or by
field experiments where the individual monoter-
pene was also tested in conjunction with high
release rates of a-pinene-containing turpentine
from the host P. taeda (Billings 1985). Further work
in this system is necessary. Recently, Poland et al.
(2003, 2004) concluded that trans-verbenol is an
aggregation pheromone component for immigrant
North American populations of T. piniperda and
that ())-a-pinene, attractive by itself, is also a host-
produced co-attractant with trans-verbenol. Byers
(2004) has hypothesized that monoterpenes may
also regulate proximal behavior of bark beetles;
specifically, to enhance entry rates into already
initiated galleries. Similar to the instances of long-
range attraction noted above, the proximal activity
of monoterpenes in this case would be in the con-
text of the aggregation pheromone emanating from
the bark surface or from the gallery itself. Thus, in
contrast to the views of early workers in the field,
who considered monoterpenes as ‘‘replaceable’’ in
the phenomenon of bark beetle aggregation
(Renwick 1970), a review of the modern literature
shows that for some species they appear to be
essential as co-attractants.
Nearly all research on the effect of monoterp-
enes as attractants or as bark beetle pheromone
co-attractants has bypassed the procedure of
sequential fractionation and assay of oleoresin
volatiles that might reveal potential synergisms
and the behavioral activity of minor components
(see Silverstein et al. 1967 for the methodology;
Silverstein 1970a, b; Byers et al. 1985; Hobson
et al. 1993 for attempts at the application).
Instead, the majority of studies have presented
beetles in the field with individual synthetic
monoterpenes or simple blends based on the most
abundant monoterpenes in host pine oleoresin.
Most recently, the selection of which compounds
to test has been guided by antennal responses in
combined gas chromatography-electroantenno-
graphic detection (GC-EAD) (e.g., Pureswaran
et al. 2004a). However, this approach has perhaps
prematurely removed the key monoterpenes from
the context of the quantitatively and qualitatively
complete odor of wound oleoresin from the pine
hosts. In a debate over the experimental approach
used to isolate and identify monoterpenes that
enhanced the response of D. brevicomis to its
pheromone, Bedard et al. (1970) wrote, ‘‘There is
no logic whatever in the a priori assumption
favoring a ‘predominant’ [quotation marks of
Bedard et al. (1970)] component over a minor
one.’’ Indeed 35 years later, it is very intriguing
that certain monoterpenes that are relatively
minor components of the volatile fraction of the
oleoresin of pine hosts play a major role in the
attraction of certain bark beetle species that col-
onize those hosts. For example, myrcene occurs
as 7% (P. ponderosa, Hobson et al. 1993), 1.4–
15.4% (P. ponderosa, Smith 1977), 20.3–20.7%
(P. coulteri, Smith 1967, 2000), 3.9% [Sierra
Nevada lodgepole pine, P. contorta murrayana
(Balfour) Critchfield, Smith 1964], 2.6% [Rocky
Mountain lodgepole pine, P. contorta latifolia
(Engelmann) Critchfield, Pureswaran et al.
2004b], 1.9–3.9% (both subspecies of P. contorta,
Smith 1983, 2000), and 4.4% (limber pine,
P. flexilis James, Zavarin et al. 1993) of the
monoterpenes in extracted oleoresin, xylem, or
152 Phytochem Rev (2006) 5:143–178
123
combined outer bark, phloem, and xylem. Yet
myrcene appears to be the most efficacious
co-attractant for the pheromone of D. brevicomis
(Wood 1972; Bedard et al. 1980), which colonizes
P. ponderosa and P. coulteri, and for the phero-
mone of D. ponderosae (Billings et al. 1976;
Miller and Borden 2000; Miller and Lindgren
2000; Pureswaran and Borden 2005), which can
colonize all of the above hosts. Terpinolene,
which is generally present in even lower quanti-
ties than myrcene in the pines noted above, is also
a highly effective co-attractant for D. ponderosae
in the Cascade Mountain (Billings et al. 1976) and
central and southern Rocky Mountain regions
(Seybold et al. unpublished data) of the western
United States (US). Pureswaran (2003) has spec-
ulated that with D. ponderosae the response to
myrcene as a pheromone co-attractant may be a
vestigial behavioral trait that reflects an earlier,
more prominent association with hosts that pro-
duced more myrcene (e.g., whitebark pine, Pinus
albicaulus Engelmann or its progenitor). Pre-
sumably, similar evolutionary hypotheses could
be posited for D. brevicomis and myrcene, and
D. ponderosae and terpinolene as well.
Some studies have evaluated the role of mon-
oterpenes as behavioral chemicals for bark bee-
tles in a more natural context. In a tree-baiting
study in Dalarna, Sweden, Schroeder and Eid-
mann (1987) found that 14-cm diameter Scots
pine, Pinus sylvestris L., trees were colonized at
significantly higher rates by T. piniperda when the
trees were baited for one day with ())-a-pinene,
(+)-3-carene, terpinolene, or the combination of
all three monoterpenes (each released at an esti-
mated 5 ll/h). In a similar study in British
Columbia, Canada with D. ponderosae, Borden
et al. (1990) reported that P. contorta latifolia
were colonized whether or not myrcene was
included in the inciting bait of female-produced
trans-verbenol and male-produced exo-brevico-
min. Presumably, myrcene or other monoterp-
enes volatilizing naturally from oleoresin released
from the newly infested trees replaced the need
for myrcene in the synthetic attractant. Pureswa-
ran and Borden (2005) also attempted to evaluate
the co-attractant role of myrcene for D. ponder-
osae in a more natural context. They reported
that the addition of myrcene (95 mg/day)
enhanced the flight response of D. ponderosae to
its aggregation pheromone more than a blend of
the five most abundant monoterpenes in P. con-
torta latifolia stem volatiles (which did not contain
myrcene). Myrcene as a co-attractant with trans-
verbenol for D. ponderosae was also numerically
(but not statistically) more efficacious than a
blend of six P. contorta latifolia monoterpenes in
funnel trapping (Conn et al. 1983) and baited tree
(Borden et al. 1983) studies.
Finally, there is a semantic issue related to the
role that host-derived monoterpenes play relative
to bark beetle aggregation pheromones in the
ensemble of attractive semiochemicals. A phero-
mone is defined as ‘‘a substance secreted by an
animal to the outside that causes a specific reaction
in another member or members of the same spe-
cies’’ (Nordlund and Lewis 1976). When a bark
beetle colonizes a pine, monoterpenes can be
emitted from wounded tree tissue or oleoresin
flowing from the wound, from boring dust that
passes around the beetle during excavation, from
undigested tree tissue in fecal material that passes
through the alimentary canal of the beetle, and
from potentially sequestered host monoterpenes
that are re-released by the beetle. Not all of these
cases are congruent with the phrase ‘secreted by an
animal’, so whether a monoterpene emanating
from a colonization site is a kairomone or an
aggregation pheromone component is a matter of
debate (Silverstein 1977; Browne et al. 1979; Bor-
den 1985). The recent discovery of a monoterpene
synthase enzyme activity in male I. pini (Martin
et al. 2003) with the implication that bark beetles
may indeed biosynthesize monoterpenes may ulti-
mately resolve this nomenclatural dilemma in cer-
tain species. Whatever functional designator we
assign to the attractive monoterpenes that are
newly released during bark beetle colonization, in
the forest airspace they join the background flux of
monoterpenes that has originated from foliage and
to a lesser extent from unwounded outer bark be-
fore and during colonization.
Monoterpenes as behavioral interruptants
Monoterpenes may also have negative conse-
quences for the survival and reproduction of pine
bark beetles. In some instances, and often at high
Phytochem Rev (2006) 5:143–178 153
123
release rates (approx. 100–2,000 mg/day), monot-
erpenes act as repellents (interruptants) to reduce
the flight responses to other behavioral chemicals
(Miller and Borden 1990a, b, 2000, 2003; Hobson
et al. 1993; Byers et al. 2000; El-Sayed and Byers
2000; Erbilgin and Raffa 2000; Erbilgin et al.
2003). Although the release rates were not
explicitly stated, Hobson et al. (1993) demon-
strated that the addition of 0.33 equivalent of (S)-
())-a-pinene (an estimated 4,125 mg/day) to one
equivalent of attractive (R)-(+)-a-pinene (an
estimated 12,375 mg/day), significantly reduced
the flight response of D. valens, providing an
example of stereospecific interruption of one
monoterpene by another (see below). In British
Columbia, terpinolene (approx. 340–2,100 mg/
day) and myrcene (approx. 60–1,300 mg/day)
interrupted the flight responses of I. latidens and I.
pini to their respective pheromones; terpinolene
(approx. 2,100 mg/day) did the same for D. pon-
derosae (Miller and Borden 2000).
As is the case with the attractive effects of
monoterpenes, little is known of the interruptive
effects in the quantitative and qualitative context of
the complete odor of wound oleoresin from an in-
fested pine. It is first perhaps of interest to ask
whether monoterpene release rates on the level of
thousands of mg are biologically relevant for trees
in pine ecosystems. Most attempts to quantify
monoterpene release rates from woody branches or
stems of pines have, for simplicity, involved small
cut logs [e.g., Browne et al. 1979, 24.2 mg/day for
myrcene from cut logs of P. ponderosa
(75 cm · 25 cm); Byers et al. 1985, 30 mg/day
for individual monoterpenes from cut logs of
P. sylvestris (28 · 13 cm); Pureswaran et al. 2004b,
10–1,200 lg/g dry tissue for individual monoterp-
enes in P. contorta latifolia] or bark chips [e.g.,
Byers et al. 2000, 48lg/day to 3.84 mg/day from P.
sylvestris or Fettig et al. 2006, 10 mg/day to 55 mg/
day from whole chipped trees from P. ponderosa
(in both cases the quantities eluted depended on
the type of monoterpene)]. These lower end esti-
mates and the likely higher release rates of mon-
oterpenes from larger sections of fallen trees, large
stump cross sections, and standing large trees
characteristic of western North American forests
suggest that monoterpenes are released from pine
tissue in nature at rates that match or exceed those
that have interrupted the flight of beetles experi-
mentally. Indeed, in a study of volatiles released
from three to five m of the main stem of P. pon-
derosa during colonization by several hundred D.
brevicomis in the Sierra Nevada of California
(Madera County), Browne et al. (1979) found that
two trees released myrcene at 50.4–112.8 mg/day/m
stem length, respectively.
It is also interesting to consider whether or not
the attractive olfactory stimuli provided by mon-
oterpenes that have functioned in behavioral
trapping assays as important attractants or pher-
omone co-attractants (but are released as minor
components of wound oleoresin) could be
drowned out in the natural context by the
cacophony of more abundant, interruptive mon-
oterpenes. Dendroctonus valens was highly
attracted in flight to a distillation fraction pre-
sumably containing most of the monoterpenes in
the oleoresin of P. ponderosa, even though the
relative abundance of an interruptant [(S)-())-a-
pinene, 14.3%] exceeded that of one of the
principal attractants [(R)-(+)-a-pinene, 0.9%]
(Hobson et al. 1993). Apparently the presence of
two other attractants [(S)-())-b-pinene, 35.8%
and (S)-(+)-3-carene, 34.4%] overcomes the
interruptive stimulus in the oleoresin. It is
tempting to hypothesize that the high release rate
interruptive effects of monoterpenes may simply
reflect an experimental artifact, i.e., generic bio-
logical or behavioral saturation at artificially high
levels (e.g., see parabolic response curve for I.
pini to racemic a-pinene in Erbilgin et al. 2003).
However, the interruptive effects depend on the
type (species) of monoterpene, and Miller and
Borden (2000) show that in I. pini and D. pon-
derosae some monoterpene co-attractants con-
tinue to elicit increasingly attractive responses,
even at extremely high release rates.
The synchrony and relevance of interruption of
flight behavior by higher release rates of certain
monoterpenes with the various phases of host
colonization (DL Wood 1982) is also poorly
understood. If long-range interruption occurs
soon after the bark is ruptured by invading bee-
tles and early in the concentration phase of host
colonization, when high density intraspecific
competition is not a factor and mates are left
unjoined, then the interruptive signals may have a
154 Phytochem Rev (2006) 5:143–178
123
net negative impact on beetle survival and
reproduction. If interruption occurs later during
the establishment phase of colonization and dis-
persing beetles are re-directed to alternative hosts
where the phloem is less fully occupied, then the
opposite impact may pertain. Interruption of
proximal host selection behavior of bark beetles
during the selection and concentration phases
may also be regulated by host monoterpenes.
From a laboratory assay, Wallin and Raffa (2000)
concluded that as concentrations of ())-, (+)-, and
(±)-a-pinene, (±)-b-pinene, and (±)-limonene
increased in the assay medium, initial gallery
entry of male I. pini decreased, the beetles were
more likely to move from amended to non-
amended portions of the medium, and gallery
length decreased. The male responses of host
entry and gallery length extension to a-pinene
were heritable traits (Wallin et al. 2002).
Monoterpenes as behavioral chemicals
for predators of pine bark beetles
Monoterpenes also influence the behavior of
insects that prey on pine bark beetles, providing
an indirect impact on the survival and reproduc-
tion of the scolytids. In this instance the pine bark
beetle herbivores occur in the middle of a tri-
trophic ‘‘sandwich’’ between the plants and the
carnivores, and the semiochemical signals move
freely across the trophic levels. The documented
effects on predators involve monoterpenes alone
and as co-attractants with bark beetle phero-
mones (i.e., multicomponent kairomones with
components derived from both of the lower tro-
phic levels). In one of the first reported cases
where monoterpenes alone elicited a flight re-
sponse from the carnivores, Rice (1969) noted
that two voracious predators of California pine
bark beetles, Temnochila chlorodia (Mann.)
(Coleoptera: Trogositidae) and Enoclerus lecontei
(Wolc.) (Coleoptera: Cleridae), responded to
a- or b-pinene in uncontrolled experiments in
which the data were not analyzed statistically.
These effects need to be re-examined using
modern methodology. With the checkered beetle,
Thanasimus dubius (F.) (Coleoptera: Cleridae), a
key predator of D. frontalis in P. taeda in the
southeastern US, Mizell et al. (1984) reported
that the predator responded in a dose-dependent
manner in a laboratory flight assay to a- and
b-pinene, both of which occur in P. taeda tur-
pentine. In a field assay, Billings (1985) found that
Temnochila virescens (F.) responded significantly
in flight to P. taeda turpentine. In several tests of a
blend of monoterpenes representative of the
Pinaceae occurring in eastern Canada, Chenier
and Philogene (1989) found that the checkered
beetles, T. dubius, Enoclerus nigripes rufiventris
(Spinola), and E. nigrifrons gerhardi Wolcott
responded significantly, although in low numbers,
to the full blend of monoterpenes (with and
without ethanol) and generally to treatments
containing (±)-a-pinene. The Eurasian predator,
Thanasimus formicarius (L.), responded at sig-
nificantly higher levels in flight to ())-a-pinene
relative to an unbaited trap (Schroeder 1988;
Schroeder and Lindelow 1989), and at signifi-
cantly higher levels to the combination of ())-a-
pinene and ethanol relative to both an unbaited
trap and to the aggregation pheromone of Ips
typographus (L.) (Schroeder 2003). Another
group of predaceous beetles, the dead log beetles
(Coleoptera: Rhizophagidae = Monotomidae),
appear to be variously attracted to ())-a-pinene
[Rhizophagus depressus (F.)] or ())-a-pinene and
ethanol [R. ferrugineus (Payk.)] (Schroeder 1988;
Schroeder and Lindelow 1989).
In many instances, monoterpenes enhance the
responses of pine bark beetle predators to the
pheromones of the herbivores. Billings (1985)
reported that P. taeda turpentine significantly in-
creased both the flight response of T. virescens to
a generic bait for Ips spp. (in two experiments)
and of Thanasimus dubius to the D. frontalis
attractant, frontalure. The flight responses of
T. dubius to pheromone components of various
pine-infesting Ips spp. were also increased sig-
nificantly by ())-a-pinene, (+)-a-pinene, and
3-carene in a series of studies in Wisconsin in the
Great Lakes Region of the US (Erbilgin and
Raffa 2001). The response of T. dubius to the
I. pini aggregation pheromone (ipsdienol and
lanierone) in Wisconsin was significantly and
dose-dependently enhanced by the addition of
racemic a-pinene (Erbilgin et al. 2003). In British
Columbia, the responses of less aggressive pre-
daceous beetles such as the wrinkled bark beetle,
Phytochem Rev (2006) 5:143–178 155
123
Lasconotus complex LeConte (Coleoptera:
Colydiidae), and darkling beetles, Corticeus sp.
Piller and Mitterpacher (Coleoptera: Tenebrion-
idae), to the kairomone ipsdienol have also been
enhanced significantly by the addition of 3-carene
or b-phellandrene or c-terpinene, and 3-carene or
b-phellandrene or a- or b-pinene, respectively
(Miller and Borden 1990a, 2000, 2003). Another
checkered beetle, T. undatulus (Say), responded
at an increased level to ipsdienol in these studies
when 3-carene supplemented the bait (Miller and
Borden 2003). In Wisconsin, ())-a-, (+)-a-, and
())-b-pinene increased responses of the preda-
ceous hister beetle, Platysoma cylindrica (Pay-
kull) (Coleoptera: Histeridae), to various
pheromone components of Ips spp., whereas ())-
and (+)-a-pinene increased responses of Corticeus
parallelus (Melsh) (Erbilgin and Raffa 2001).
Interestingly, in several instances in these exper-
iments the monoterpene myrcene interrupted the
response of T. dubius to Ips spp. pheromone
components (Erbilgin and Raffa 2001), poten-
tially representing a net beneficial impact on the
herbivore from the presence of this monoterpene
in the semiochemical message.
Monoterpenes, pine defenses, and effects
on bark beetle physiology
Monoterpenes are also detrimental physiologi-
cally to pine bark beetles as a consequence of their
role in defense of pines. Defense of these long-
lived trees consists of anatomical and chemical
components that are both constitutive and induc-
ible (Nebeker et al. 1993; Langenheim 2003;
Franceschi et al. 2005). Pines have vertical and
horizontal interconnected resin canal systems that
span both the xylem and the phloem (Langenheim
2003). As a consequence, pines defend themselves
against breaches in their outer bark by bark bee-
tles and other invaders to a greater degree from
their constitutive or preformed defenses than they
do from their induced defenses (Nebeker et al.
1993). Further, sapling pines have a high level of
monoterpene cyclase (monoterpene synthase)
activity in the constitutive resin canal system that
does not increase significantly upon wounding of
the stem (Lewinsohn et al. 1991). Whether this
biochemical effect holds for larger trees typically
colonized by bark beetles remains to be estab-
lished. Treatment of P. contorta latifolia and
P. taeda with bark beetle-associated fungi results
in hypersensitive response lesions whose oleoresin
appears to contain quantitatively and qualitatively
different monoterpene compositions than con-
stitutive oleoresin (Shrimpton 1973; Raffa and
Berryman 1982, 1983; Paine et al. 1987; reviewed
in Nebeker et al. 1993).
When the outer bark is opened, the defense
system of pines manifests itself in both physical
and chemical terms through the release of oleo-
resin from severed resin canals. Stark (1965)
defined oleoresin as ‘‘...the non-aqueous secretion
of resin acids dissolved in a terpene hydrocarbon
oil which is (a) produced in or exuded from the
intercellular resin ducts of a living tree; ......’’ For
example, when D. ponderosae colonizes P. pon-
derosa, P. contorta latifolia, or other species, the
first few pioneers are often killed or driven out by
the mass flow of oleoresin that emanates from the
resin canal system and pours out the nascent en-
trance tunnel (Beal 1939). This is especially evi-
dent if the host tree has adequate moisture and
oleoresin exudation pressure (Stark 1965).
Blackman (1931) described the elaborate behav-
ior of female D. ponderosae during the early
stages of colonization. The female alternatively
bites tree tissue from the phloem–xylem interface
and retreats frequently to the outside surface of
the bark where she spreads and disposes of masses
of oleoresin adhering to her body. This historical
description underscores the lengthy contact peri-
od during which female Dendroctonus spp. are
exposed to the physical obstacle presented by
oleoresin as well as its potentially toxic hydro-
carbons (Nebeker et al. 1993). Since insects take
oxygen into their bodies through pleural spiracles
(lateral aperatures) along the thorax and abdo-
men, immersion of bark beetles in oleoresin may
have a suffocating as well as a toxic effect. Hodges
et al. (1977, 1979) reported that the resistance of
four native pine species in the southeastern US to
colonization by D. frontalis was strongly related
through a discriminant analysis to physical prop-
erties of the oleoresin such as total flow, flow rate,
viscosity, and time to crystallization.
The role of monoterpenes in the chemical
defense of pines rests on the experimental
156 Phytochem Rev (2006) 5:143–178
123
evidence that upon prolonged exposure at close
range, monoterpenes can be insecticidal to pine
bark beetles (Smith 1961, 1965a, b; Cook and
Hain 1988, Table 1). Specifically, at high doses in
closed containers, they exhibit a fumigant toxicity
effect (Smith 1961, 1965a, b; Byers et al. 1979;
Byers 1981; Cook and Hain 1988). Byers et al.
(1979) reported that after an 18 h exposure, the
percentage of ‘‘comatose’’ male California fives-
pined ips, Ips paraconfusus Lanier, increased
sharply when the headspace concentration of
myrcene in a sealed glass bottle reached approx.
4 lg/ml. Similar studies with a-pinene resulted in
mortality in the 40–50% range when the head-
space concentration reached approx. 18 lg/ml
(Byers 1981). Byers and Birgersson (1990) esti-
mated that the vapor concentration of myrcene in
an I. paraconfusus nuptial chamber in P. pon-
derosa was 0.028 lg/ml. Thus, whether the vola-
tile insecticidal effects measured in closed
containers in the laboratory pertain in the more
open system of a gallery whose volatiles are ex-
hausted by ventilation through an entrance hole,
or perhaps through the somewhat porous bark
surface, has yet to be examined experimentally.
However, given the descriptions of Blackman
(1931) and Beal (1939) noted above, Dendroct-
onus spp. adults may come in prolonged contact
with high concentrations of monoterpenes dis-
solved in liquid oleoresin during attempts at host
colonization. Smith (1966) reported that even
brief immersion of D. brevicomis adults in fresh
resin had a deleterious effect on ability to feed
subsequently in pine phloem, and resins of non-
host pines increased the rate of mortality of the
adults relative to resin of P. ponderosa.
The influence of oxygenated monoterpenes
on pine bark beetles
In addition to large-scale emissions of monot-
erpenes sensu stricto from vegetation in pine
forests, there is a growing realization that most
monoterpenes emitted in these forests may un-
dergo rapid oxidation through exposure to fre-
quently encountered atmospheric oxidants such
as hydroxyl (OH)) and nitrate (NO3) radicals,
and ozone (O3) (Atkinson and Arey 2003; Holz-
inger et al. 2005b; Lee et al. 2006). These land-
scape-level oxidation products may influence the
host colonization behavior of pine bark beetles,
but appear upon first analysis (AH Goldstein,
personal communication) to be of much smaller
molecular weight than most oxygenated monot-
erpenes that elicit behavioral responses from bark
beetles (Seybold et al. 2000).
Oxygenated monoterpenes and pheromone
biosynthesis
Monoterpene oxidation also occurs on a more
localized scale, driven by biological rather than
physical chemical processes. Perhaps the most
intimate relationship between pines, their mon-
oterpenes, and pine bark beetles is the
involvement of the isoprenoids in pheromone
biosynthesis by the beetles. In male I. para-
confusus, the ())-enantiomer of a-pinene is
converted to cis-verbenol (Fig. 2) (Renwick
et al. 1976a), a key component of the three-part
aggregation pheromone (Silverstein et al. 1966).
Another monoterpene, myrcene, is converted
into the other two pheromone components,
ipsdienol (2-methyl-6-methylene-2,7-octadien-4-
ol) and ipsenol (2-methyl-6-methylene-7-octen-
4-ol), by this species (Byers et al. 1979; Hendry
et al. 1980). Similar conversions of monoterp-
enes to behaviorally active oxygenated com-
pounds also occur in other coniferophagous
species (Hughes 1973a, b, 1974, 1975; Renwick
et al. 1973, 1976b; Klimetzek and Francke 1980;
Byers 1982, 1983a, b; Hunt et al. 1986; Pierce
et al. 1987; Hunt and Smirle 1988; Lindstrom
et al. 1989; Gries et al. 1990; Vanderwel 1991;
Seybold et al. 1992; Barkawi 2002). In addition
to enzymatic transformations endogenous to
bark beetles, other potential sources of behav-
iorally active oxygenated monoterpenes include
autoxidation (Hunt et al. 1989; Grosman 1996)
and conversions that are mediated by bacteria
or fungi that are symbiotic with the beetles
(Brand et al. 1975, 1976; Byers and Wood 1981;
Conn et al. 1984; Hunt and Borden 1989a, b).
In all of these cases, the origin of these mon-
oterpenes has been thought to be the oleoresin
associated with the phloem or outer xylem in
pines or other conifers.
Phytochem Rev (2006) 5:143–178 157
123
Over the last 10 years the research on biosyn-
thesis of pine bark beetle aggregation phero-
mones has shifted the focus to de novo pathways
present endogenously in the beetles (Seybold
et al. 1995; Ivarsson et al. 1997; Tillman et al.
1998, 2004; Barkawi et al. 2003). It has become
clear that evolution has provided bark beetles
with an elaborate mechanism for self-contained
synthesis of these critically important coloniza-
tion and reproductive signals to guide their
assemblages (Seybold and Tittiger 2003). For
example, male I. pini synthesize ipsdienol de novo
through the regulatory control of juvenile hor-
mone (JH III), which appears to act primarily on
HMG-CoA reductase in the mevalonate (MVA)
pathway (Tillman et al. 2004). Multiple enzymes
in this pathway are upregulated during phero-
mone biosynthesis in several bark beetle species
(Tillman et al. 1998; Tittiger et al. 2000; Martin
et al. 2003; Gilg et al. 2005), and gene expression
for these enzymes is coordinated (Keeling et al.
2004). With I. pini, cell-free extracts of male tis-
sue will also convert geranyl diphosphate (GDP)
to the monoterpene myrcene in a regulated
fashion, providing the first biochemical evidence
for a monoterpene synthase in the Metazoa
(Martin et al. 2003), and explaining successful
pheromone biosynthesis in Pinus spp. that appear
to contain insufficient quantities of available host
myrcene (Byers and Birgersson 1990). Tissue
from female I. pini does not carry out this con-
version. The synthesis of myrcene is stimulated by
both feeding on host pine phloem and treatment
with JH III, which are both correlates of phero-
mone biosynthesis.
In biochemical terms, there is a rather
remarkable nexus of the 2-C-methyl-D-erythritol-
4-phosphate (MEP) pathway in pines with the
MVA pathway in pine bark beetles (Fig. 3). The
pathways overlap when the pines and beetles
convert isomeric dimethylallyl diphosphate and
isopentyl diphosphate to GDP; they are joined
when the beetles utilize myrcene from the host
and/or de novo synthesized myrcene to form the
pheromone alcohol endproducts. Thus, phero-
mone synthesized from pine-based myrcene
originates from the MEP pathway, whereas
pheromone synthesized from beetle-based myr-
cene originates from the MVA pathway.
Oxygenated monoterpenes and cytochrome
P450s
In the last stages of pheromone biosynthesis, the
monoterpene alcohol and ketone pheromone end
products in pine bark beetles are likely formed
through the catalytic activity of cytochrome P450
enzymes (P450s) (White et al. 1979, 1980; Hunt
and Smirle 1988). These enzymes may form
enantiospecific oxygenated products from pro-
chiral monoterpenes (e.g., myrcene) or from chi-
ral monoterpenes (e.g., a- or b-pinene). P450s
occur ubiquitously in organisms ranging from
bacteria to fungi to plants to animals (Omura
1999). In eukaryotes, they catalyze NADPH-
dependant oxidations on an extremely diverse
array of substrates. In animals, they are involved
in detoxification of plant secondary metabolites,
hormone biosynthesis and degradation, phero-
mone biosynthesis and degradation, and metab-
olism of fatty acids (Feyereisen 1999; Omura
1999).
There have been only a few studies directly
targeting P450-related physiology or biochemistry
of pine bark beetles. White et al. (1979) found that
microsomes isolated from larval and adult black
turpentine beetles, Dendroctonus terebrans (Oli-
vier), converted a-pinene to a-pinene oxide and
other oxidation products. Further, they reported
that although a-pinene induced cytochrome P450
activity in rat liver microsomes, it did not do so in
D. terebrans microsomes. In experiments with
D. ponderosae, females and males treated with the
P450 inhibitor, piperonyl butoxide, yielded
abdominal extracts that displayed a reduced con-
version of a-pinene and myrcene to trans-verbenol
and ipsdienol, respectively, as well as an accumu-
lation of the monoterpene precursors (Hunt and
Smirle 1988). The biosynthesis of exo-brevicomin
by male D. ponderosae involves the incorporation
of molecular oxygen during the epoxidation of
(Z)-6-nonen-2-one (Vanderwel and Oehlschlager
1992), and this reaction is likely catalyzed by a
P450. Also using D. ponderosae as a model, Pierce
et al. (1987) outlined the pathways for P. pon-
derosa and P. contorta latifolia monoterpene
metabolism through oxygenation (Fig. 4). The
conversions involve mainly allylic hydroxylation
and hydration reactions focused on double bonds
158 Phytochem Rev (2006) 5:143–178
123
in the carbon skeleton; epoxidation reactions may
also occur. These allylic hydroxylations and ep-
oxidations, which likely involve molecular oxygen,
serve as a prelude to the isolation and character-
ization of P450s from pine bark beetles by illus-
trating the scope of functionalities necessary for
beetles during host colonization. For example, as
noted above, in I. paraconfusus and other Ips spp.
it is likely that the final or penultimate biosyn-
thetic reaction in pheromone production, the
conversion of myrcene to ipsdienol, is catalyzed by
a P450 (Fig. 4C). Because in many cases the final
pheromone product consists of blends of both (+)-
and ())-ipsdienol, it is possible that two separate
P450s catalyze the enantiospecific reactions.
In an attempt to find the P450s potentially in-
volved in pheromone biosynthesis in I. paracon-
fusus, and to set a foundation for a deeper
understanding of the plethora of events during
bark beetle colonization of host tissue, we have
used degenerate PCR techniques to identify and
clone 14 P450s from cDNA derived from RNA
isolated from male and female I. paraconfusus fed
in P. ponderosa phloem for 20 h. Further rapid
amplification of cDNA ends has allowed the iso-
lation of full length cDNAs of eight of the 14
P450s. We are continuing work on obtaining full
length cDNAs for the other six P450s for use in
functional characterization of the enzymatic
activity of their protein products. Other insects
whose genomes have been more extensively
characterized (e.g., the fruit fly, Drosophila mel-
anogaster Meigen and the mosquito, Anopheles
gambiae Giles) have approx. 100 P450 genes
(Adams et al. 2000; Gomez et al. 2005). Since
endophytic pine bark beetles have an intimate
interaction with a plant host defense system, we
might anticipate that they have at least as many, if
not more, P450 genes than these Diptera. Thus,
we have isolated perhaps 10 to 15% of the
ensemble of P450s present in I. paraconfusus.
All but two of the 14 P450s seem to belong to
the Cyp4 family of P450s, the most common
subfamily of insect P450s. One is a member of the
Fig. 3 Proposed interaction of monoterpenoid biosynthet-ic pathways in pines and pine bark beetles showingdifferent origins of C5 units from the 2-C-methyl-D-erythritol-4- phosphate (MEP) and mevalonate (MVA)pathways for the convergent synthesis of myrcene.
Myrcene is oxidized to ipsdienol (likely by P450s) in pinebark beetles. Figure reproduced in modified form fromFig. 1 on page 174 in Martin et al. (2003) with kindpermission of Springer Science and Business Media
Phytochem Rev (2006) 5:143–178 159
123
160 Phytochem Rev (2006) 5:143–178
123
Cyp9 family; another is a member of the Cyp31
family and is likely not of insect origin (i.e., con-
tamination from nematodes or mites, which are
internally and externally phoretic, respectively,
with pine bark beetles, Kinn 1971; Massey 1974;
Stephen et al. 1993). All 14 P450s were subjected
to quantitative, real-time PCR-based expression
analyses. Individual male and female I. paracon-
fusus were fed for 0, 8, or 24 h in fresh P. pon-
derosa phloem. Each sex and time point was
represented by 12 individual insects (i.e., 12 rep-
licates, each consisting of one insect). Following
feeding, HMG-CoA reductase transcript levels
[used as a control because the expression pattern
of this gene in I. paraconfusus is well character-
ized (Tillman et al. 2004)] increased dramatically
in males fed for 8 and 24 h, but as expected did
not change in females that do not produce pher-
omone, providing evidence that the insects had
responded appropriately to their exposure to host
tissue. In addition, 10 of the 14 P450s showed
statistically significant differential transcript
accumulation in males and/or in females, usually,
but not always, within 8 h of initial contact with
host phloem. The Cyp31 gene was among the four
that did not show differential transcript accumu-
lation in either sex following feeding. The differ-
ential expression responses that we have observed
may be classified into six groups.
First, we observed three genes that were
upregulated in males, but whose expression levels
did not change in females. We hypothesize that
these may be involved in pheromone biosynthesis
(a potential substrate is myrcene), male-specific
juvenile hormone (JH) biosynthesis (Feyereisen
et al. 1981) (potential substrates are methyl far-
nesoate or farnesoic acid), or detoxification of
constitutive defenses encountered by pioneering
males making the first encounter with a host tree
(potential substrates include various terpenoids
or plant ecdysteroids).
Second, we observed some genes that were
downregulated in males, but did not change in
females. Genes of this class would include genes
that were no longer required, or whose expression
would be detrimental for males that had suc-
cessfully located and colonized a pine host. We
hypothesize that these genes may be involved in
degradation of pheromones (Wojtasek and Leal
1999; Maıbeche-Coisne et al. 2004) or host kair-
omones that directed the insects to the tree in the
first place (potential substrates would include
pheromone components or host compounds that
are behaviorally-active to foraging bark beetles).
Upon arriving and colonizing a suitable pine host,
male behavior may be altered if persistent for-
aging-related signals were received and pro-
cessed. In addition, after males arrive at a host
and begin to feed, their juvenile hormone titers
should increase (Tillman et al. 1998). Thus genes
that degrade JH may also be downregulated at
this point. Such enzymes would likely have JH,
methyl farnesoate, farnesoic acid, farnesol, or
farnesal as substrates (Sutherland et al. 1998).
Third, we observed P450s that were upregu-
lated in both sexes after feeding. These could be
involved in cis-verbenol biosynthesis, as it is
produced by both sexes (Byers 1981, 1983b). The
most likely substrate in this case would be
a-pinene. The generality of this transformation of
a-pinene is illustrated by its widespread occur-
rence in nature, ranging from bacteria and fungi
(Brand et al. 1975; Prema and Bhattacharyya
1962) to human tissues (Eriksson and Levin
1990); the latter followed by the excretion of the
conjugated alcohol in the urine. Because both
sexes of I. paraconfusus are confronted with tox-
ins from pines, and because both sexes require
high titers of JH during host colonization, this
class of P450s might be involved in xenobiotic
detoxification or JH biosynthesis. Flight muscle
degradation, which begins in both sexes of
I. paraconfusus immediately after host coloniza-
tion (Borden and Slater 1969; Bhakthan et al.
1970), and is stimulated by JH (Borden and Slater
1968; Unnithan and Nair 1977), is another process
Fig. 4 Theoretical oxidative transformations of monot-erpenes from ponderosa pine, Pinus ponderosa, and RockyMountain lodgepole pine, Pinus contorta latifolia (Engel-mann) Critchfield by the mountain pine beetle, Dendroct-onus ponderosae Hopkins (modified from Pierce et al.1987). (A) Allylic oxidation and rearrangement productsof ())-a-pinene; (B) allylic oxidation and hydrationproducts of ())-b-pinene; (C) allylic oxidation and hydra-tion products of myrcene; (D) allylic oxidation andrearrangement products of (+)-3-carene; and (E) allylicoxidation and hydration products of ())-b-phellandrene.Numerical identification of structures as in Pierce et al.(1987)
b
Phytochem Rev (2006) 5:143–178 161
123
that might involve P450s upregulated in both
sexes following feeding. In addition, reproductive
activity likely increases metabolic requirements
dramatically, and thus fatty acids may be a sub-
strate for these enzymes (Aoyama et al. 1990;
Feyereisen 1999; Omura 1999). One P450 in our
study, the Cyp9 family gene, showed upregulation
after feeding on the order of almost 105· in males
but only 102· in females, both compared to non-
fed insects. A P450 involved in myrcene detoxi-
fication and in conversion of myrcene to ipsdienol
might show such a pattern in that males would
need to rapidly clear myrcene that had been
synthesized in the midgut, as ipsdienol, both to
allow survival (clearance of a toxin) and to attract
mates and conspecific males, whereas phloem-
mining females would have to detoxify the tree-
produced myrcene. Thus, in such a situation, both
males and females might produce transcripts of
the same gene, but at different levels reflecting
the different roles played by the protein product
of the gene in host colonization and reproductive-
related activity.
Fourth, we observed P450s that were down-
regulated in both sexes after feeding. As with the
P450s that were downregulated in males only
after feeding, these may be involved in degrada-
tion of behaviorally active chemicals in the
antennae or in degradation of JH.
Fifth, we observed a gene that was downregu-
lated in males but upregulated in females. This
gene may be involved in female-specific JH or
ecdysone production (Tillman-Wall et al. 1992;
Blomquist et al. 1994) in preparation for repro-
ductive activity, with possible substrates including
methyl farnesoate, farnesoic acid, or a number of
candidates from the ecdysone biosynthesis path-
way (Warren et al. 2002). Alternately, this gene
may be involved in ‘‘heavy duty’’ detoxification of
host secondary metabolites. Because the female is
the later-arriving sex, and she carries out more
extensive boring activity in the phloem than the
male, she may be confronted with constitutive or
fungally stimulated induced defense responses
that differ in quantity and quality from those
presented to the male. Thus, females are likely
assaulted at the site of infestation with particu-
larly toxic secondary metabolites in large quan-
tities, and they may express a special ensemble of
P450s that is able to deal with such major threats
to their reproductive success.
Finally, we observed some genes that were
constitutively expressed in both sexes at what
seem to be high levels at all time points before
and after feeding. For example, because we
detected a consistent signal for one of the P450
genes in over 98.6% of all samples regardless of
sex or feeding status, it was chosen as the
housekeeping gene for the quantitative analysis of
expression. This and similar genes could be
involved in constitutive detoxification of host
secondary metabolites or basic and relatively
continuous metabolic processes, e.g., fatty acid
metabolism. The functions of such constitutively
expressed genes could be highly varied and will
possibly be quite difficult to predict.
The primary amino acid sequences of P450s do
not provide information that allows precise pre-
diction of their function. Thus, while our work to
date has set a firm foundation for the study of
P450s in bark beetles, further research will
require functional characterization of each of the
P450s that we have thus far cloned from I. para-
confusus. Functional characterization, combined
with further expression analyses of these and
other P450s following treatment of the insects
with hormones, plant secondary metabolites, or at
different insect life stages, will provide a much
better understanding of the important events just
prior to and following host colonization by these
ecologically- and economically important insects.
Oxygenated monoterpenes and stereospecific
responses by pine bark beetles
The enantiomeric composition of kairomone and
pheromone components of pine bark beetles is a
critical determinant of behavioral activity (Wood
et al. 1976; Birch et al. 1980; Francke and Vite
1983; Francke et al. 1986; Seybold 1993). With
D. valens, Hobson et al. (1993) clearly showed a
strong preference in flight response to the kairo-
mone (R)-(+)-a-pinene; the antipode interrupted
the response to the (+)-enantiomer. Strangely, the
enantiomeric composition of a-pinene in the
oleoresin of P. ponderosa, one of the primary
hosts in this region, was 95%-()). With oxygen-
ated monoterpene pheromones, perhaps the best
162 Phytochem Rev (2006) 5:143–178
123
example involves western populations of I. pini.
Birch et al. (1980) found that ethanol solutions of
(R)-())-ipsdienol were attractive in the lab and
field, whereas solutions of (S)-(+)-ipsdienol were
interruptive. As little as 5–10% of the (+)-enan-
tiomer caused a significant reduction in trap catch
in response to the ())-enantiomer. Below we dis-
cuss two current projects in our laboratory in
which the flight responses of I. paraconfusus and
the Mediterranean pine engraver, Orthotomicus
(Ips) erosus (Wollaston), are governed by the
enantiomeric composition of the oxygenated
monoterpene pheromone components.
Ips paraconfusus
The California fivespined ips is an important and
polyphagous pest of pines in Oregon and Cali-
fornia (Struble and Hall 1955; Schultz and Bedard
1987). Its broad host range and capacity to thrive
in coastal as well as montane climates make it a
potential for concern as an invasive species in
other parts of North America and other conti-
nents. It is very abundant on adventive plantings
of Monterey pine, Pinus radiata D. Don, in urban
landscapes in coastal California, and P. radiata is
the most widely planted pine in the world with
plantations covering nearly 4 million ha in
southern hemisphere locations such as Australia,
Chile, New Zealand, and South Africa (Lavery
and Mead 1998). Thus, an efficacious aggregation
pheromone bait for I. paraconfusus would be an
important detection tool for international pest
management programs.
As noted above, the male-produced phero-
mone of I. paraconfusus is a synergistic blend of
three monoterpene alcohols, ipsenol, ipsdienol,
and cis-verbenol (Silverstein et al. 1966; Wood
et al. 1967, 1968). The predominant naturally
occurring enantiomers isolated from males were
(4S)-())-ipsenol, (4S)-(+)-ipsdienol, and (1S,2S)-
(+)-cis-verbenol, which occurred in a ratio of
100:10:2 (Wood et al. 1967). The optical rotation
of cis-verbenol varies depending on the solvent in
which it is measured [acetone or methanol,
(1S,2S)-(+); chloroform, (1S,2S)-())]. Although
Silverstein et al. (1966) reported the original
natural product as [a]D21 = +4�, measured in
acetone, most literature subsequent to Mori et al.
(1976) and commercial vendors refer to (1S,2S)-
cis-verbenol as the ())-enantiomer, i.e., as mea-
sured in chloroform. The commercially available
pheromone for I. paraconfusus is an equal (race-
mic) mixture of the optical isomers of ipsenol
(220 lg/day), a highly-enriched blend (approx.
97%) of (+)-ipsdienol (110 lg/day), and 83%-
(1S,2S)-())-cis-verbenol (300–600 lg/day) (Phero
Tech Inc., Delta, British Columbia, Canada, all
release rates measured at 25�C) (Fig. 2). Thus,
the stereochemistry of the components of the
commercially available pheromone matches, in
part, the naturally occurring compounds; the rel-
ative release rates do not match the naturally
occurring component ratios.
In 2004 and 2005 we used multiple funnel traps
and pheromone components from Phero Tech
Inc. and ChemTica Internacionale S.A. (Heredia,
Costa Rica) in modern release devices to test the
preference of I. paraconfusus for the various
enantiomers in three sequential experiments at
the University of California, Blodgett Research
Forest in El Dorado Co., California (Table 2).
This was the site of the historic first field study of
this pheromone system in June of 1966 (Wood
et al. 1967). Treatments were organized in a
randomized complete block design of four blocks,
and checked and re-randomized every few days
(nine, seven, and thirteen times in experiments 1–
3, respectively). In experiment 1, the enantiomers
of ipsdienol [97%-(+) and 97%-())] were tested
in combination with racemic ipsenol and 83%-
())-cis-verbenol. The experiment also included
conophthorin, a spiroacetal that is known to
interrupt the flight response of other species of
Ips (Huber et al. 2001; Zhang 2003). Conoph-
thorin has been isolated from a wide range of
natural sources, including cone beetles, twig
beetles, wasps, and angiosperm tree bark (Huber
et al. 1999, 2000; Francke and Kitching 2001;
Zhang and Schlyter, 2004).
We found that I. paraconfusus had a strong
preference for the bait containing (+)-ipsdienol
(Table 2). A 2· release rate of racemic ipsdienol
attracted fewer I. paraconfusus than the 1· re-
lease rate of (+)-ipsdienol; this indicates that the
())-enantiomer of ipsdienol interrupts the attrac-
tive response, confirming previous California
Phytochem Rev (2006) 5:143–178 163
123
Ta
ble
2P
rog
ress
ion
of
ex
pe
rim
en
tsto
de
mo
nst
rate
the
en
an
tio
spe
cifi
cre
spo
nse
of
the
Ca
lifo
rnia
fiv
esp
ine
dip
s,Ip
sp
ara
con
fusu
s,to
ph
ero
mo
ne
com
po
ne
nts
,B
lod
ge
ttF
ore
stR
ese
arc
hS
tati
on
,E
lD
ora
do
Co
.,C
ali
forn
ia,
20
04
–2
00
5(S
ey
bo
lde
ta
l.u
np
ub
lish
ed
da
ta)
Ex
pe
rim
en
tD
ate
sG
oa
lsT
rea
tme
nts
aO
utc
om
es
12
7A
ug
ust
–2
2S
ep
tem
be
r,2
00
4O
pti
miz
eth
ee
na
nti
om
eri
cco
mp
osi
tio
no
fip
sdie
no
lb,
c,
d
Ipsd
ien
ol
inv
ari
ou
sb
len
ds
[ra
cem
ic1·
and2
·,(+
)-1·,
())-1·]
whilekeeping
83%
-())-
cis-verbenolandracemic
ipse-
nolconstantin
each
treatm
ent,conoph-
thorin
alone,
conophthorin
added
to(+
)-ipsdienol&
cis-verbenol&
ipsenol,
unbaited
trap(7
treatm
ents)
Tre
atm
en
tw
ith
(+)-
ips-
die
no
la
ttra
ctiv
e;
wit
h()
)-ip
sdie
no
ln
ot
att
rac-
tiv
e;
race
mic
1·
and
2·
partiallyattractivedueto
interruptionby())-ipsdie-
nol;
conophthorin
inter-
ruptive
Te
stin
terr
up
tio
nb
yco
no
ph
tho
rin
22
8Ju
ly–
12
Au
gu
st,
20
05
Op
tim
ize
the
en
an
tio
me
ric
com
po
siti
on
of
cis-
ve
rbe
no
l
cis-
Ve
rbe
no
lin
ble
nd
s[(
+),
()),
an
d8
3%
-()
)]o
ra
bse
nt
wh
ile
ke
ep
ing
(+)-
ipsd
ien
ol
an
dra
cem
icip
sen
ol
con
sta
nt
ine
ach
tre
atm
en
t,u
nb
ait
ed
tra
p(5
)
Tre
atm
en
tsw
ith
())-
cis-
ve
rbe
no
la
ttra
ctiv
e;
wit
h(+
)-ci
s-v
erb
en
ol
we
ak
lya
ttra
ctiv
e;
hig
he
rre
-sp
on
seto
83
%-(
))-
cis-
ve
rbe
no
lv
s.()
)-ci
s-v
er-
be
no
ld
ue
toa
hig
he
rre
lea
sera
te3
12
Au
gu
st–
19
Se
pte
mb
er,
20
05
Op
tim
ize
the
en
an
tio
me
ric
com
po
siti
on
of
ipse
no
lb,
d
Ipse
no
lin
ble
nd
s[(
+)-
1·,
())-1
·,race-
mic
1·and2
·]orabsentwhilekeeping
(+)-ipsdienoland
83%
-())-
cis-verbe-
nolconstantin
each
treatm
ent,unbait-
edtrap(6)
Tre
atm
en
tsw
ith
())-
ipse
-n
ol
att
ract
ive
;w
ith
(+)-
ipse
no
ln
ot
att
ract
ive
,ra
cem
ic2·
ismost
eco-
nomical
and
effective
form
of
ipsenol
inthe
experim
ent
Fu
ture
dir
ect
ion
s2
00
6T
est
va
ryin
gra
tio
so
fip
sdie
no
l,ci
s-v
erb
en
ol
an
dip
sen
ol
Ipsd
ien
ol,
cis-
ve
rbe
no
l,ip
sen
ol
in1
0:2
:10
0ra
tio
tom
imic
na
tura
lp
he
ro-
mo
ne
,a
nd
inv
ary
ing
com
po
ne
nt
rati
os,
un
ba
ite
dtr
ap
Te
stth
ee
ffe
cto
fa-
pin
en
eT
hre
e-p
art
ble
nd
alo
ne
an
dco
mb
ine
dw
ith
com
me
rcia
lly
av
ail
ab
lea-pinene,
andracemic
2·,
(+)-1·,
())-1
·blends,
unbaited
trap(6)
aA
llm
ate
ria
lsfr
om
Ph
ero
Te
ch,
Inc.
un
less
oth
erw
ise
ind
ica
ted
.F
or
all
en
an
tio
me
ric
mix
ture
so
fip
sdie
no
l,1
·re
lea
sera
tes
are
0.1
1m
g/d
ay
.8
3%
-()
)-ci
s-V
erb
en
ol
rele
ase
rate
is0
.3–
0.6
mg
/da
ya
t2
5�C
,a
nd
(+)-
an
d()
)-ci
s-v
erb
en
ol
rate
sa
re0
.08
mg
/da
ya
t2
0�C
(Ch
em
Tic
a,
Inte
rna
cio
na
leS
.A.)
.F
or
all
en
an
tio
me
ric
mix
ture
so
fip
sen
ol,
1·
rele
ase
rate
sa
re0
.22
–0
.24
mg
/da
y.
Ra
cem
icco
no
ph
tho
rin
rele
ase
rate
is3
.0m
g/d
ay
.b
Lig
ht
an
dB
irch
(19
79
).c
Pa
ine
an
dH
an
lon
(19
91
).d
Ko
hn
lee
ta
l.(1
99
4).
164 Phytochem Rev (2006) 5:143–178
123
studies from Siskyou Co. (Light and Birch 1979),
San Diego Co. (Paine and Hanlon 1991), and
Nevada Co. (Kohnle et al. 1994). Conophthorin
also interrupted the response of I. paraconfusus to
the attractant blend containing (+)-ipsdienol,
suggesting that in addition to aiding in avoiding
non-host angiosperms (Huber et al. 1999, 2000;
Zhang and Schlyter, 2004), it may also aid in
maintaining species specificity in pheromone
communication with pine-infesting cone and twig
beetles that use it as a pheromone component
(Birgersson et al. 1995; Pierce et al. 1995; Dallara
et al. 2000).
In experiment 2, various enantiomeric blends
of cis-verbenol were tested in combination with
(+)-ipsdienol and racemic ipsenol. The blend with
())-cis-verbenol was highly attractive to I. para-
confusus, whereas the blend with (+)-cis-verbenol
was only weakly attractive and not different from
the two-component blend without any cis-verbe-
nol (Table 2). We observed differences in flight
responses to ())-cis-verbenol from the two com-
mercial vendors that are likely due to the sub-
stantial differences in the release rates of the
formulations (Phero Tech: 300–600 lg/day at
25�C versus ChemTica: 80 lg/day at 20�C). Since
an enantiomeric blend of cis-verbenol that con-
tained 17% of the (+)-enantiomer was quite
attractive, (+)-cis-verbenol is likely not interrup-
tive. However, this needs to be confirmed with a
trial comparing responses to racemic cis-verbenol
released at 1· and 2· with responses to (+)- and
())-cis-verbenol released at 1·. A review of the
literature reveals that no previous studies have
attempted to determine the impact of the enan-
tiomeric composition of cis-verbenol on the flight
response of I. paraconfusus.
In experiment 3, the enantiomers of ipsenol
[97%-(+) and 97%-())] were tested in combina-
tion with (+)-ipsdienol and 83%-())-cis-verbenol.
The blend with ())-ipsenol attracted I. paracon-
fusus, whereas the blend with (+)-ipsenol did not
(Table 2). Since the response to the 2· release rate
of racemic ipsenol was similar to the response to
the 1· release rate of ())-ipsenol, (+)-ipsenol is
likely not interruptive. Our results suggest that a
higher release rate of racemic ipsenol relative to
(+)-ipsdienol would be a more efficacious attrac-
tant for I. paraconfusus. Ten release devices of the
currently available formulation of racemic ipsenol
to one of (+)-ipsdienol [1100:110 lg/day, ())-
ipsenol:(+)-ipsdienol] would most accurately align
the synthetic bait with the naturally occurring
component ratios. A review of the literature re-
vealed only one study that investigated the impact
of the enantiomeric composition of ipsenol on the
flight response of I. paraconfusus. Light and Birch
(1979) reported that in Siskiyou Co. (+)-ipsenol
did not reduce the flight response to a P. pon-
derosa log infested with male I. paraconfusus (i.e.,
the naturally produced aggregation pheromone),
and this is consistent with the results of our
experiment 3. The sexes responded in the same
patterns for all treatments and experiments out-
lined above.
Future research on the enantiospecific
response of I. paraconfusus to its three-compo-
nent pheromone blend will involve a more con-
trolled study of the impact of the enantiomeric
composition of cis-verbenol (see above); a study
that varies the individual components in tandem
and separately, and a study that investigates the
role of the enantiomers of a-pinene and perhaps
other monoterpenes as co-attractants.
Orthotomicus erosus
The invasion of exotic species of plants and ani-
mals has led to major ecological and economic
problems (Pimentel et al. 2000). From an insect
pest management perspective, worldwide com-
merce and transport of wood packing and plant
materials are resulting in the homogenization of
the bark beetle fauna across international borders
(Wood and Bright 1992). In a 15-year survey,
many scolytids have been intercepted in barked
rough wood associated with packing materials
that carry tiles, marble, machinery and other
construction goods to US ports (Haack 2001,
2006). This growing problem is especially notable
in California where the number of established
exotic bark beetle species has doubled to nearly
20 species in the last few years (Penrose et al. in
preparation).
The discovery of the Mediterranean pine
engraver, Orthotomicus erosus, in California in
May of 2004 (JC Lee et al. 2005) is an example of
one new invasive species that raises serious
Phytochem Rev (2006) 5:143–178 165
123
concerns. It is a generalist pest of pines in its
native range in the Mediterranean, Middle East,
and Central Asia, and in introduced areas of
Chile, Fiji, and South Africa (Eglitis 2000). Gen-
erally, O. erosus infests standing pine trees under
stress, recently fallen trees, broken branches or
logging debris. Besides causing feeding damage,
O. erosus has vectored some ophiostomoid fungi
that are pathogenic to pines (Wingfield and
Marasas 1980). In the southern Central Valley of
California, this beetle has been found infesting
pine trees and cut logs in parks, golf courses, and
other urban landscapes.
The chemical ecology of O. erosus has been
studied in Europe, Israel, and South Africa.
Giesen et al. (1984) used combined gas chroma-
tography–mass spectrometry to analyze the
headspace gas from hindguts dissected from male
O. erosus that had infested logs of maritime pine,
Pinus maritima Lamarck (=P. pinaster Ait.). The
chemical analysis and subsequent field test in
South Africa confirmed that ipsdienol and 2-me-
thyl-3-buten-2-ol (MB) (Fig. 2) were major com-
ponents of the aggregation pheromone. The
combination of ipsdienol and MB was necessary
to attract O. erosus; traps baited with ipsdienol
alone (Giesen et al. 1984; Serez 1987) or MB
alone (Klimetzek and Vite 1986; Mendel 1988)
attracted few beetles. These authors suggested
that ipsdienol was a long-distance signal, whereas
MB influenced landing behavior of O. erosus.
Other common bark beetle pheromone compo-
nents, such as ipsenol, cis-verbenol, trans-verbe-
nol, and frontalin did not appear to influence the
flight behavior of O. erosus near Bordeaux,
France (Klimetzek and Vite 1986). At a field site
near Lisbon, Portugal, verbenone and possibly
cis-verbenol inhibited O. erosus attraction to
ipsdienol and MB (Paiva et al. 1988). The release
rates (Klimetzek and Vite 1986) and enantio-
meric composition (Kohnle 1991) of ipsdienol
were studied for their effect on O. erosus flight
response, but the results were inconclusive. The
impact of the release rate of MB on the response
of O. erosus has not been evaluated previously.
Therefore, release rates, enantiomeric composi-
tion, and the effect of host monoterpene co-attr-
actants (e.g., a-pinene) are all research questions
that need to be addressed to optimize the
attractant bait to improve detection of this beetle
in North America.
In a series of experiments in 2005 in Fresno and
Tulare Cos., California, we tested the flight
response of O. erosus to pheromone and host
compounds using baited multiple funnel traps
(Table 3). Treatments were organized in a ran-
domized complete block design of four blocks, and
checked and re-randomized once or twice every
week. In experiment 1, responses were evaluated
to racemic ipsdienol, MB, and ())-a-pinene, alone
and in combination. Orthotomicus erosus
responded at very low levels to each of the com-
ponents alone, but responded synergistically to
racemic ipsdienol and MB (Table 3). Experiments
2 and 3 optimized the release rates of MB and
racemic ipsdienol, respectively. The results of
experiment 4 indicated that beetles were strongly
attracted to ())-ipsdienol, whereas (+)-ipsdienol
was interruptive, making the racemic blend of
ipsdienol inappropriate for an optimal attractant.
Results of experiment 5 confirmed that O. erosus
responded synergistically to ())-ipsdienol and MB.
The efficacy of the bait was proven in experiments
4 and 5 where the responses to ())-ipsdienol and
MB exceeded the responses to male pheromone
produced naturally in small cut logs of Aleppo
pine, Pinus halepensis Miller, each containing 25
feeding males. The sexes responded in the same
patterns for all treatments and experiments.
Future research on the enantiospecific
response of O. erosus to its two-component
pheromone blend will involve a study that varies
the release rate of ())-ipsdienol; a study that
varies the release rates of ())-ipsdienol and MB
in tandem; and a study that investigates the role
of the enantiomers of a-pinene as co-attractants.
In experiment 1, the role of ())-a-pinene was not
clear because only one trap on one date had
excessively high captures in the bait containing
racemic ipsdienol, MB, and ())-a-pinene
(Table 3).
2-Methyl-3-buten-2-ol has been shown to be a
major volatile released by pine needles (foliage)
from ten species of pines from western North
America, including seven that occur in California
(Harley et al. 1998). Fluxes of oxygenated volatile
organic compounds above a P. ponderosa plan-
tation in California were dominated by MB and
166 Phytochem Rev (2006) 5:143–178
123
Ta
ble
3P
rog
ress
ion
of
ex
pe
rim
en
tsto
de
mo
nst
rate
the
en
an
tio
spe
cifi
cre
spo
nse
of
the
Me
dit
err
an
ea
np
ine
en
gra
ve
r,O
rth
oto
mic
us
ero
sus,
tov
ari
ou
sp
he
rom
on
ea
nd
ho
stco
mp
ou
nd
sin
Fre
sno
an
dT
ula
reC
os.
,C
ali
forn
ia,
20
05
(Le
ee
ta
l.u
np
ub
lish
ed
da
ta)
Ex
pe
rim
en
tD
ate
sG
oa
lsT
rea
tme
nts
aO
utc
om
es
17
Fe
bru
ary
–1
8M
arc
h,
20
05
Te
stsy
ne
rgis
mo
f2
-me
thy
l-3
-b
ute
n-2
-ol
(MB
),ra
cem
icip
sdie
no
l,a
nd
a-pinene
b,c
MB
,ra
cem
icip
sdie
no
l,a
nd
a-pinenealoneandin
combination,
unbaited
trap(6
treatm
ents)
MB
&ra
cem
icip
sdie
no
lsy
ne
rgis
tic;
ino
ne
case
MB
&ra
c.ip
sdie
no
l&
a-pineneattractive
22
8M
arc
h–
15
July
,2
00
5O
pti
miz
ere
lea
sera
teo
fM
BM
Ba
t0
.5–
1.8
,1
7–
60
,8
1–
27
1,
81
0–
27
10
mg
/da
yw
hil
ek
ee
pin
gra
cem
icip
sdie
no
lre
lea
seco
nst
an
tin
ea
chtr
ea
tme
nt,
un
ba
ite
dtr
ap
(5)
MB
rele
ase
rate
of
0.5
–6
0m
g/d
ay
&ra
cem
icip
s-d
ien
ol
mo
sta
ttra
ctiv
e
31
5Ju
ly–
2S
ep
tem
be
r,2
00
5O
pti
miz
ere
lea
sera
teo
fra
cem
icip
sdie
no
lcR
ace
mic
ipsd
ien
ol
at
0.1
1,
0.5
5,
an
d5
.55
mg
/da
yw
hil
ek
ee
pin
gM
Bco
n-
sta
nt
ine
ach
tre
atm
en
t,u
nb
ait
ed
tra
p(4
)
No
dif
fere
nce
inre
spo
nse
tolo
wa
nd
hig
hip
sdie
no
lre
lea
sera
tes
&M
B;
low
rele
ase
de
vic
em
ore
eco
-n
om
ica
l4
2–
23
Se
pte
mb
er,
20
05
Op
tim
ize
en
an
tio
me
ric
com
po
-si
tio
no
fip
sdie
no
ldIp
sdie
no
lin
ble
nd
s[r
ace
mic
1·and
2·,
(+)-1·,
())-1·]
while
keeping
MB
constant
ineach
treatm
ent,
male-infested
log,unbaited
trap(6)
())-
Ipsd
ien
ol
&M
Ba
ttra
ctiv
ea
nd
the
com
bi-
na
tio
no
utp
erf
orm
sn
atu
-ra
lp
he
rom
on
e;
race
mic
1·and2
·partiallyattrac-
tivedueto
interruptionby
(+)-ipsdienol
Co
mp
are
ton
atu
ral
ph
ero
mo
ne
of
25
ma
les
52
3S
ep
tem
be
r–1
8O
cto
be
r,2
00
5T
est
syn
erg
ism
of
())-
ipsd
ien
ol
an
dM
BM
B,
())-
ipsd
ien
ol
alo
ne
an
din
com
bin
ati
on
,m
ale
-in
fest
ed
log
,u
n-
ba
ite
dtr
ap
(4)
())-
Ipsd
ien
ol
&M
Bsy
n-
erg
isti
ca
nd
the
com
bin
a-
tio
no
utp
erf
orm
sn
atu
ral
ph
ero
mo
ne
Co
mp
are
ton
atu
ral
ph
ero
mo
ne
of
25
ma
les
Fu
ture
dir
ect
ion
s2
00
6O
pti
miz
ere
lea
sera
teo
f()
)-ip
sdie
no
l()
)-Ip
sdie
no
la
tfo
ur
rele
ase
rate
sw
hil
ek
ee
pin
gM
Bco
nst
an
t,u
nb
a-
ite
dtr
ap
(5)
Te
ste
ffe
cto
fa-
pin
en
e()
)-Ip
sdie
no
l&
MB
alo
ne
an
dco
mb
ine
dw
ith
com
me
rcia
lly
av
ail
-a
ble
,ra
cem
ic2·,
(+)-1·,
())-1·-
a-pinene,
unbaited
trap(6)
aA
llm
ate
ria
lsfr
om
Ph
ero
Te
ch,
Inc.
un
less
oth
erw
ise
ind
ica
ted
.F
or
all
en
an
tio
me
ric
mix
ture
so
fip
sdie
no
l,re
lea
sera
tes
are
0.1
1m
g/d
ay
un
less
oth
erw
ise
spe
cifi
ed
.M
Bre
lea
sera
tes
are
17
–6
0m
g/d
ay
un
less
oth
erw
ise
spe
cifi
ed
.MB
fro
mS
igm
aA
ldri
chw
as
de
liv
ere
dfr
om
40
0ll
pla
stic
Ep
pe
nd
orf
tub
es
top
rod
uce
are
lea
sera
teo
f0
.5–
1.8
,a
nd
fro
m1
5m
lp
last
icb
ott
les
top
rod
uce
rele
ase
rate
so
f8
1–
27
1,
an
d8
10
–2
71
0(a
llm
g/d
ay
).C
om
me
rcia
lly
av
ail
ab
le9
5%
-()
)-a-
pin
en
ew
as
rele
ase
dfr
om
15
ml
pla
stic
bo
ttle
sa
t1
50
mg
/da
ya
t2
3�C
.bG
iese
ne
ta
l.(1
98
4);
Me
nd
el
(19
88
).cK
lim
etz
ek
an
dV
ite
(19
86
).dK
oh
nle
(19
91
).
Phytochem Rev (2006) 5:143–178 167
123
methanol (ca. 1.3 mg C m)2 h)1) (Schade and
Goldstein 2001). Thus, an intriguing possibility
with O. erosus in California is that the high emis-
sion rates (>5 lg C g)1 h)1) of MB from the foli-
age of P. contorta murrayana, P. coulteri, Jeffrey
pine, P. jeffreyi Balfour, P. ponderosa, Bishop
pine, P. muricata D. Don, gray pine, P. sabiniana
Dougl. ex D. Don, and Torrey pine, P. torreyana
Parry ex Carr., may play a role in the chemical
ecology of this immigrant bark beetle species
(Harley et al. 1998). Pinus coulteri
(70.6 lg C g)1 h)1), P. sabiniana (67 lg C
g)1 h)1), and P. torreyana (37.3 lg C g)1h)1),
whose native populations are all distributed in
relative proximity to the introduced population of
O. erosus, had particularly high emission rates of
MB (Harley et al. 1998). Widely planted P. radi-
ata had an intermediate emission rate of this
hemiterpenoid in the survey. Although MB re-
leased from foliage may be an ecologically inap-
propriate context for host colonization by O.
erosus, the high vapor phase concentrations of MB
in forests containing these hosts may serve as a
general attractant for O. erosus, specifically in in-
stances when ipsdienol-producing native Ips spp.
(e.g., I. latidens, I. spinifer (Eichhoff), I. mexicanus
(Hopkins), I. plastographus maritimus Lanier,
I. pini, or I. paraconfusus) are colonizing the
branches or main stems of these trees. The
behavioral activity of MB has been tested recently
in the mixed conifer forest of California’s Sierra
Nevada (Gray 2002), prior to the potential inva-
sion of this forest by O. erosus. In that study, two
native, pine-infesting species, I. paraconfusus and
D. brevicomis, and their common predators
(beetles in the families Trogositidae and Cleridae
that were not determined to species) did not ap-
pear to respond significantly to MB as an attrac-
tive or interruptive signal.
The introduction of O. erosus into California
also provides an opportunity to study the bio-
synthesis of MB in a pine-infesting bark beetle
species and provides new motivation to study the
formation of MB in pine host trees (Fig. 5). This
hemiterpenoid is a relatively unusual and infre-
quently occurring pheromone structure among
the bark beetles (Seybold and Vanderwel 2003).
Its biosynthesis in bark beetles has been studied
briefly in the Eurasian spruce engraver, Ips
typographus L. (Lanne et al. 1989), but nothing is
known of how the biosynthesis of MB is regulated
in bark beetles or what role, if any, terpene syn-
thases or P450s may play in the conversion of
dimethylallyl diphosphate (DMAPP) to the
alcohol endproduct. In contrast, regulation of the
formation of MB has been studied to some extent
in pines. For example, in P. ponderosa needle
tissue the formation of MB occurs via the MEP
pathway (Zeidler and Lichtenthaler 2001). While
it is possible that P450s could be involved in the
oxidation of DMAPP in pine bark beetles, there
is evidence from research with needle tissue of
P. sabiniana that MB is formed instead by a ter-
pene synthase enzyme activity (Fisher et al.
2000). The reaction mechanism of pine MB syn-
thase in the formation of MB from DMAPP could
be similar to the formation of the monoterpene
alcohol linalool from GDP in Norway spruce,
Dimethylallyl- Isopentenyl-diphosphate
H3CCCH2CH2O
H2Cdiphosphate
H3CC CHCH2O
H3C
PP PP
2-Methyl-3-buten-2-ol
H3C
C CH2H3C
OH
MEP or MVA
?
H
C
Fig. 5 Biosynthesis of the hemiterpenoid 2-methyl-3-buten-2-ol (MB) has not been completely elucidated inbark beetles, but likely involves modifications of dimeth-ylallyl diphosphate (DMAPP) or isopentenyl diphosphate(IDP), either of which could be derived from either the2-C-methyl-D-erythritol-4-phosphate (MEP) or mevalo-nate (MVA) pathway. Based on initial labeling studies(Lanne et al. 1989), the latter pathway is the more likelyroute to 2-methyl-3-buten-2-ol in bark beetles. In Pinusponderosa, MB is derived from the MEP pathway (Zeidlerand Lichtenthaler 2001)
168 Phytochem Rev (2006) 5:143–178
123
Picea abies L. Karst, and other plant species
(Martin et al. 2004). The role of a terpene syn-
thase in the formation of the related hemiterpene
isoprene from DMAPP is also well established in
plants (Miller et al. 2001). In the case of the for-
mation of MB from DMAPP the reactive carbo-
cation intermediate in the terpene synthase
reaction would be quenched by the addition of
water, instead of proton elimination as occurs in
the formation of isoprene.
Conclusions
Pine bark beetles are significant forest pests with
an interesting reproductive biology that is guided
in many cases by host monoterpenes and iso-
prenoid aggregation pheromones. In a few spe-
cies, host monoterpenes are attractive alone as
long-range signals, but they have been recognized
repeatedly in many species as essential co-attr-
actants with aggregation pheromones. The mon-
oterpenes arise in the pines via the MEP pathway.
Some pheromones can arise both from host
monoterpenes and through de novo synthesis in
the beetles via the MVA pathway. Both produc-
tion of pheromones and flight response to pher-
omones are stereospecific processes. Research
currently underway on bark beetle pheromone
biosynthesis will broaden our understanding of
the role of P450’s in stereospecific oxygenation
reactions of monoterpenes and in hemiterpenoid
biosynthesis. Ongoing research on stereospecific
responses of I. paraconfusus and O. erosus will
optimize the efficacy of commercial baits to de-
tect these species as potential invaders in ports
and forested regions in other continents (I. para-
confusus) and within North America (O. erosus).
Acknowledgements We gratefully acknowledge theHuman Frontier Science Program (Grant #RGY0382) forsupport of collaborative research conducted by theSeybold and Bohlmann Laboratories. We also thank threeanonymous reviewers for their critical contributions to themanuscript; C. Leutenegger and T. Olineka, Lucy WhittierMolecular and Diagnostic Core Facility at UC-Davis, andM. Erickson, USDA Forest Service, PSW, for assistancewith molecular expression analyses; K. Daane, Universityof California at Berkeley, F. Schurr and S. Rambeau,University of California Blodgett Forest Research Station,R. West, Valley Oaks Golf Course, Visalia, California, and
E. Espiritu, K. Gandhi, S. Hamud, P. Jiros, J. Lacsina, andO. Singh (all USDA Forest Service, PSW) for assistancewith field studies; and J.A. Tillman for assistance withgraphics.
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